Receptor activator of NF-κB

ABSTRACT

Isolated receptors, DNAs encoding such receptors, and pharmaceutical compositions made therefrom, are disclosed. The isolated receptors can be used to regulate an immune response. The receptors are also useful in screening for inhibitors thereof.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/996,139filed Dec. 22, 1997 U.S. Pat. No. 6,017,729, which is acontinuation-in-part of U.S. Ser. No. 60/064,671, filed Oct. 14, 1997,and a continuation in part of U.S. Ser. No. 08/813,509, filed Mar. 7,1997 (converted to provisional application U.S. Ser. No. 60/077,181, onAug. 18, 1997) and a continuation-in part of U.S. Ser. No. 08/772,330,filed Dec. 23, 1996 (converted to provisional application U.S. Ser. No.60/059,978, on Jul. 24, 1997).

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of cytokinereceptors, and more specifically to cytokine receptor/ligand pairshaving immunoregulatory activity.

BACKGROUND OF THE INVENTION

Efficient functioning of the immune system requires a fine balancebetween cell proliferation and differentiation and cell death, to ensurethat the immune system is capable of reacting to foreign, but not selfantigens. Integral to the process of regulating the immune andinflammatory response are variuos members of the Tumor Necrosis Factor(TNF) Receptor/Nerve Growth Factor Receptor Superfamily (Smith et al.,Science 248:1019; 1990). This family of receptors includes two differentTNF receptors (Type I and Type II Smith et al., supra; and Schall etal., Cell 61:361, 1990), nerve growth factor receptor Johnson et al.,Cell 47:545, 1986), B cell antigen CD40 (Stamenkovic et al., EMBO J.8:1403, 1989), CD27 (Camerini et al., J. Immunol. 147:3165, 1991), CD30(Durkop et al., Cell 68:421, 1992) T cell antigen OX40 (Mallett et al.,EMBO J. 9:1063, 1990), human Fas antigen (Itoh et al., Cell 66:233,1991), murine 4-1BB receptor (Kwon et al., Proc. Natl. Acad Sci. USA86:1963, 1989) and a receptor referred to as Apoptosis-Inducing Receptor(AIR; U.S. Ser. No. 08/720,864, filed Oct. 4, 1996).

CD40 is a receptor present on B lymphocytes, epithelial cells and somecarcinoma cell lines that interacts with a ligand found on activated Tcells, CD40L (U.S. Ser. No. 08/249,189, filed May 24, 1994). Theinteraction of this ligand/receptor pair is essential for both thecellular and humoral immune response. Signal transduction via CD40 ismediated through the association of the cytoplasmic domain of thismolecule with members of the TNF receptor-associated factors (TRAFs;Baker and Reddy, Oncogene 12:1, 1996). It has recently been found thatmice that are defective in TRAF3 expression due to a targeted disruptionin the gene encoding TRAF3 appear normal at birth but developprogressive hypoglycemia and depletion of peripheral white cells, anddie by about ten days of age (Xu et al., Immunity 5:407, 1996). Theimmune responses of chimeric mice reconstituted with TRAF3^(−/−) fetalliver cells resemble those of CD40-deficient mice, although TRAF3^(−/−)B cells appear to be functionally normal.

The critical role of TRAF3 in signal transduction may be in itsinteraction with one of the other members of the TNF receptorsuperfamily, for example, CD30 or CD27, which are present on T cells.Alternatively, there may be other, as yet unidentified members of thisfamily of receptors that interact with TRAF3 and play an important rolein postnatal development as well as in the development of a competentimmune system. Identifying additional members of the TNF receptorsuperfamily would provide an additional means of regulating the immuneand inflammatory response, as well as potentially providing furtherinsight into post-natal development in mammals.

SUMMARY OF THE INVENTION

The present invention provides a novel receptor, referred to as RANK(for receptor activator of NF-κB), that is a member of the TNF receptorsuperfamily. RANK is a Type I transmembrane protein having 616 aminoacid residues that interacts with TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6.Triggering of RANK by over-expression, co-expression of RANK andmembrane bound RANK ligand (RANKL), and with addition of soluble RANKLor agonistic antibodies to RANK results in the upregulation of thetranscription factor NF-κB, a ubiquitous transcription factor that ismost extensively utilized in cells of the immune system.

Soluble forms of the receptor can be prepared and used to interfere withsignal transduction through membrane-bound RANK, and hence upregulationof NF-κB; accordingly, pharmaceutical compositions comprising solubleforms of the novel receptor are also provided. Inhibition of NF-κB byRANK antagonists may be useful in ameliorating negative effects of aninflammatory response that result from triggering of RANK, for examplein treating toxic shock or sepsis, graft-versus-host reactions, acuteinflammatory reactions, and the effects of excess bone resorption.Soluble forms of the receptor will also be useful in in vitro and invivo based screening tests for agonists or antagonists of RANK activity.

The cytoplasmic domain of RANK will be useful in developing assays forinhibitors of signal transduction, for example, for screening formolecules that inhibit interaction of RANK with TRAF1, TRAF2, TRAF3,TRAF5 and in particular TRAF6. Deleted forms and fusion proteinscomprising the novel receptor are also disclosed.

The present invention also identifies a counterstructure, or ligand, forRANK, referred to as RANKL. RANKL is a Type 2 transmembrane protein withan intracellular domain of less than about 50 amino acids, atransmembrane domain and an extracellular domain of from about 240 to250 amino acids. Similar to other members of the TNF family to which itbelongs, RANKL has a ‘spacer’ region between the transmembrane domainand the receptor binding domain that is not necessary for receptorbinding. Accordingly, soluble forms of RANKL can comprise the entireextracellular domain or fragments thereof that include the receptorbinding region.

These and other aspects of the present invention will become evidentupon reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the influence of RANK.Fc and hRANKL on activated Tcell growth. Human peripheral blood T cells were cultured as describedin Example 12. Viable T cell recovery was determined by triplicatetrypan blue countings.

FIG. 2 illustrates the ability of RANKL to induce human DC clusterformation. Functionally mature dendritic cells (DC) were generated invitro from CD34⁺ bone marrow (BM) progenitors and cultured as describedin Example 13. CD1a+ DC were cultured in a cytokine cocktail alone(upper left panel), in cocktail plus CD40L (upper right), RANKL (lowerleft), or heat inactivated (ΔH) RANKL, and then photographed using aninversion microscope.

FIG. 3 demonstrates that RANKL enhances DC allo-stimulatory capacity.Allogeneic T cells were incubated with varying numbers of irradiated DCcultured as described in Example 13. The cultures were pulsed with[³H]-thymidine and the cells harvested onto glass fiber sheets forcounting. Values represent the mean±standard deviation (SD) oftriplicate cultures.

DETAILED DESCRIPTION OF THE INVENTION

A novel partial cDNA insert with a predicted open reading frame havingsome similarity to CD40 was identified in a database containing sequenceinformation from cDNAs generated from human bone marrow-deriveddendritic cells (DC). The insert was used to hybridize to colony blotsgenerated from a DC cDNA library containing full-length cDNAs. Severalcolony hybridizations were performed, and two clones (SEQ ID NOs:1 and3) were isolated. SEQ ID NO:5 shows the nucleotide and amino acidsequence of a predicted full-length protein based on alignment of theoverlapping sequences of SEQ ID NOs:1 and 3.

RANK is a member of the TNF receptor superfamily; it most closelyresembles CD40 in the extracellular region. Similar to CD40, RANKassociates with TRAF2 and TRAF3 (as determined by co-immunoprecipitationassays substantially as described by Rothe et al., Cell 83:1243, 1995).TRAFs are critically important in the regulation of the immune andinflammatory response. Through their association with various members ofthe TNF receptor superfamily, a signal is transduced to a cell. Thatsignal results in the proliferation, differentiation or apoptosis of thecell, depending on which receptor(s) is/are triggered and which TRAF(s)associate with the receptor(s); different signals can be transduced to acell via coordination of various signaling events. Thus, a signaltransduced through one member of this family may be proliferative,differentiative or apoptotic, depending on other signals beingtransduced to the cell, and/or the state of differentiation of the cell.Such exquisite regulation of this proliferative/apoptotic pathway isnecessary to develop and maintain protection against pathogens;imbalances can result in autoimmune disease.

RANK is expressed on epithelial cells, osteoclast precursors, some Bcell lines, and on activated T cells. However, its expression onactivated T cells is late, about four days after activation. This timecourse of expression coincides with the expression of Fas, a known agentof apoptosis. RANK may act as an anti-apoptotic signal, rescuing cellsthat express RANK from apoptosis as CD40 is known to do. Alternatively,RANK may confirm an apoptotic signal under the appropriatecircumstances, again similar to CD40. RANK and its ligand are likely toplay an integral role in regulation of the immune and inflammatoryresponse.

Moreover, the post-natal lethality of mice having a targeted disruptionof the TRAF3 gene demonstrates the importance of this molecule not onlyin the immune response, but also in development. The isolation of RANK,as a protein that associates with five of the 6 known TRAFs, and itsligand will allow further definition of this signaling pathway, anddevelopment of diagnostic and therapeutic modalities for use in the areaof autoimmune and/or inflammatory disease. The RANK/TRAF association canbe utilized in screening methodologies to discover therapeutics,including small molecules and peptides, that modulate RANK/TRAFinteraction and thus effect the activities that result from theinteraction. The Examples below demonstrate that TRAF6 is particularlyimportant in mediating RANK signaling. Therapeutics that inhibitRANK/TRAF6 interaction are immunosuppressants and anti-inflammatoryagents. Compounds that interfere with RANK/TRAF6 interactions are alsouseful for modulating the formation of osteoclasts from osteoclastprecursors, modulating osteoclast function and activities, and asinhibitors of diseases associated with excess bone resorption.

DNAs, Proteins and Analogs

The present invention provides isolated RANK polypeptides and analogs(or muteins) thereof having an activity exhibited by the native molecule(i.e, RANK muteins that bind specifically to a RANK ligand expressed oncells or immobilized on a surface or to RANK-specific antibodies;soluble forms thereof that inhibit RANK ligand-induced signaling throughRANK). Such proteins are substantially free of contaminating endogenousmaterials and, optionally, without associated native-patternglycosylation. Derivatives of RANK within the scope of the inventionalso include various structural forms of the primary proteins whichretain biological activity. Due to the presence of ionizable amino andcarboxyl groups, for example, a RANK protein may be in the form ofacidic or basic salts, or may be in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction. The primaryamino acid structure may be modified by forming covalent or aggregativeconjugates with other chemical moieties, such as glycosyl groups,lipids, phosphate, acetyl groups and the like, or by creating amino acidsequence mutants. Covalent derivatives are prepared by linkingparticular functional groups to amino acid side chains or at the N- orC-termini.

Derivatives of RANK may also be obtained by the action of cross-linkingagents, such as M-maleimidobenzoyl succinimide ester andN-hydroxysuccinimide, at cysteine and lysine residues. The inventiveproteins may also be covalently bound through reactive side groups tovarious insoluble substrates, such as cyanogen bromide-activated,bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activatedagarose structures, or by adsorbing to polyolefin surfaces (with orwithout glutaraldehyde cross-linking). Once bound to a substrate, theproteins may be used to selectively bind (for purposes of assay orpurification) antibodies raised against the proteins or against otherproteins which are similar to RANK or RANKL, as well as other proteinsthat bind RANK or RANKL or homologs thereof.

Soluble forms of RANK are also within the scope of the invention. Thenucleotide and predicted amino acid sequence of the RANK is shown in SEQID NOs:1 through 6. Computer analysis indicated that the protein has anN-terminal signal peptide; the predicted cleavage site follows residue24. Those skilled in the art will recognize that the actual cleavagesite may be different than that predicted by computer analysis. Thus,the N-terminal amino acid of the cleaved peptide is expected to bewithin about five amino acids on either side of the predicted, preferredcleavage site following residue 24. Moreover a soluble form beginningwith amino acid 33 was prepared; this soluble form bound RANKL. Thesignal peptide is predicted to be followed by a 188 amino acidextracellular domain, a 21 amino acid transmembrane domain, and a 383amino acid cytoplasmic tail.

Soluble RANK comprises the signal peptide and the extracellular domain(residues 1 to 213 of SEQ ID NO:6) or a fragment thereof. Alternatively,a different signal peptide can be substituted for the native leader,beginning with residue 1 and continuing through a residue selected fromthe group consisting of amino acids 24 through 33 (inclusive) of SEQ IDNO:6. Fragments of the extracellular or cytoplasmic domain can beprepared using known techniques to isolate a desired portion of thedomain region of interest. For example, one such technique includescomparing the extracellular or cytoplasmic domain with those of othermembers of the TNFR family and selecting forms similar to those preparedfor other family members. Alternatively, unique restriction sites or PCRtechniques that are known in the art can be used to prepare numeroustruncated forms which can be expressed and analyzed for activity.

Also included within the scope of the invention are fragments orderivatives of the intracellular domain of RANK. Such fragments areprepared by any of the herein-mentioned techniques, and include, but arenot limited to, peptides that are identical to the full cytoplasmicdomain of RANK as shown in SEQ ID NO:6, (amino acid 234-616) or ofmurine RANK as shown in SEQ ID NO:15, and those that comprise a portionof the cytoplasmic region. As described in Examples 19 and 20, truncatedforms of the RANK cytoplasmic domain have been identified as TRAFbinding sites. Such fragments include the COOH-terminal 72 amino acids(amino acids 545-616) which interacts with TRAFs 1, 2, 3, 5 and 6, aminoacids 339-422 which interacts with TRAF6, amino acids 339-362 whichinteracts with TRAF6, and all of SEQ ID NO:6. Similarly, fragments ofthe cytoplasmic domain of muRANK (SEQ ID NO:14 and SEQ ID NO:15) thatare conserved regions with SEQ ID NO:5 and SEQ ID NO:6, respectively,are important for TRAF binding, and encompassed by the presentinvention. Accordingly, a PCR-based technique was developed tofacilitate preparation of various C-terminal truncations that wouldretain the conserved regions.

All techniques used in preparing soluble forms may also be used inpreparing fragments or analogs of the cytoplasmic domain (i.e., RT-PCRtechniques or use of selected restriction enzymes to preparetruncations). DNAs encoding all or a fragment of the intracytoplasmicdomain will be useful in identifying other proteins that are associatedwith RANK signaling, for example using the immunoprecipitationtechniques described herein, or another technique such as a yeasttwo-hybrid system (Rothe et al., supra).

Other derivatives of the RANK proteins within the scope of thisinvention include covalent or aggregative conjugates of the proteins ortheir fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.For example, the conjugated peptide may be a signal (or leader)polypeptide sequence at the N-terminal region of the protein whichco-translationally or post-translationally directs transfer of theprotein from its site of synthesis to its site of function inside oroutside of the cell membrane or wall (e.g., the yeast α-factor leader).

Protein fusions can comprise peptides added to facilitate purification,identification, and function of RANK proteins, RANK homologs (e.g.,poly-His), RANK fragments, including fragments of the cytoplasmic domainand extracellular domain. The amino acid sequence of the inventiveproteins can also be linked to an identification peptide such as thatdescribed by Hopp et al., Bio/Technology 6:1204 (1988). Such a highlyantigenic peptide provides an epitope reversibly bound by a specificmonoclonal antibody, enabling rapid assay and facile purification ofexpressed recombinant protein. The sequence of Hopp et al. is alsospecifically cleaved by bovine mucosal enterokinase, allowing removal ofthe peptide from the purified protein. Fusion proteins capped with suchpeptides may also be resistant to intracellular degradation in E. coli.

Fusion proteins further comprise the amino acid sequence of a RANK orRANK fragment linked to an immunoglobulin Fc region. An exemplary Fcregion is a human IgG₁ having a nucleotide an amino acid sequence setforth in SEQ ID NO:8. Fragments of an Fc region may also be used, as canFc muteins. For example, certain residues within the hinge region of anFc region are critical for high affinity binding to FcγRI. Canfield andMorrison (J. Exp. Med 173:1483; 1991) reported that Leu₍₂₃₄₎ andLeu₍₂₃₅₎ were critical to high affinity binding of IgG₃ to FcγRI presenton U937 cells. Similar results were obtained by Lund et al. (J. Immunol.147:2657, 1991; Molecular Immunol. 29:53, 1991). Such mutations, aloneor in combination, can be made in an IgG₁ Fc region to decrease theaffinity of IgG₁ for FcR. Depending on the portion of the Fc regionused, a fusion protein may be expressed as a dimer, through formation ofinterchain disulfide bonds. If the fusion proteins are made with bothheavy and light chains of an antibody, it is possible to form a proteinoligomer with as many as four RANK regions.

In another embodiment, RANK proteins and RANK fragments further comprisean oligomerizing peptide such as a leucine zipper domain. Leucinezippers were originally identified in several DNA-binding proteins(Landschulz et al., Science 240:1759, 1988). Leucine zipper domain is aterm used to refer to a conserved peptide domain present in these (andother) proteins, which is responsible for dimerization of the proteins.The leucine zipper domain (also referred to herein as an oligomerizing,or oligomer-forming, domain) comprises a repetitive heptad repeat, withfour or five leucine residues interspersed with other amino acids.Examples of leucine zipper domains are those found in the yeasttranscription factor GCN4 and a heat-stable DNA-binding protein found inrat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Twonuclear transforming proteins, fos and jun, also exhibit leucine zipperdomains, as does the gene product of the murine proto-oncogene, c-myc(Landschulz et al., Science 240:1759, 1988). The products of the nuclearoncogenes fos and jun comprise leucine zipper domains preferentiallyform a heterodimer (O'Shea et al., Science 245:646, 1989; Turner andTjian, Science 243:1689, 1989). The leucine zipper domain is necessaryfor biological activity DNA binding) in these proteins.

The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess leucine zipper domains (Buckland and Wild, Nature 338:547,1989;Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research andHuman Retroviruses 6:703, 1990). The leucine zipper domains in thesefusogenic viral proteins are near the transmembrane region of theproteins; it has been suggested that the leucine zipper domains couldcontribute to the oligomeric structure of the fusogenic proteins.Oligomerization of fusogenic viral proteins is involved in fusion poreformation (Spruce et al, Proc. Nati. Acad. Sci. U.S.A 88:3523, 1991).Leucine zipper domains have also been recently reported to play a rolein oligomerization of heat-shock transcription factors (Rabindran etal., Science 259:230, 1993).

Leucine zipper domains fold as short, parallel coiled coils. (O'Shea etal., Science 254:539; 1991) The general architecture of the parallelcoiled coil has been well characterized, with a “knobs-into-holes”packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). Thedimer formed by a leucine zipper domain is stabilized by the heptadrepeat, designated (abcdefg)_(n) according to the notation of McLachlanand Stewart (J. Mol. Biol. 98:293; 1975), in which residues a and d aregenerally hydrophobic residues, with d being a leucine, which line up onthe same face of a helix. Oppositely-charged residues commonly occur atpositions g and e. Thus, in a parallel coiled coil formed from twohelical leucine zipper domains, the “knobs” formed by the hydrophobicside chains of the first helix are packed into the “holes” formedbetween the side chains of the second helix.

The leucine residues at position d contribute large hydrophobicstabilization energies, and are important for dimer formation (Krysteket al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al. recentlyreported the synthesis of a triple-stranded a-helical bundle in whichthe helices run up-up-down (Science 259:1288, 1993). Their studiesconfirmed that hydrophobic stabilization energy provides the maindriving force for the formation of coiled coils from helical monomers.These studies also indicate that electrostatic interactions contributeto the stoichiometry and geometry of coiled coils.

Several studies have indicated that conservative amino acids may besubstituted for individual leucine residues with minimal decrease in theability to dimerize; multiple changes, however, usually result in lossof this ability (Landschulz et al., Science 243:1681, 1989; Turner andTjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990). vanHeekeren et al. reported that a number of different amino residues canbe substituted for the leucine residues in the leucine zipper domain ofGCN4, and further found that some GCN4 proteins containing two leucinesubstitutions were weakly active (Nucl. Acids Res. 20:3721, 1992).Mutation of the first and second heptadic leucines of the leucine zipperdomain of the measles virus fusion protein (MVF) did not affectsyncytium formation (a measure of virally-induced cell fusion); however,mutation of all four leucine residues prevented fusion completely(Buckland et al., J. Gen. Virol. 73:1703, 1992). None of the mutationsaffected the ability of MVF to form a tetramer.

Amino acid substitutions in the a and d residues of a synthetic peptiderepresenting the GCN4 leucine zipper domain have been found to changethe oligomerization properties of the leucine zipper domain (Alber,Sixth Symposium of the Protein Society, San Diego, Ca.). When allresidues at position a are changed to isoleucine, the leucine zipperstill forms a parallel dimer. When, in addition to this change, allleucine residues at position d are also changed to isoleucine, theresultant peptide spontaneously forms a trimeric parallel coiled coil insolution. Substituting all amino acids at position d with isoleucine andat position a with leucine results in a peptide that tetramerizes.Peptides containing these substitutions are still referred to as leucinezipper domains.

The present invention also includes RANK with or without associatednative-pattern glycosylation. Proteins expressed in yeast or mammalianexpression systems, e.g., COS-7 cells, may be similar or slightlydifferent in molecular weight and glycosylation pattern than the nativemolecules, depending upon the expression system. Expression of DNAsencoding the inventive proteins in bacteria such as E. coli providesnon-glycosylated molecules. Functional mutant analogs of RANK proteinhaving inactivated N-glycosylation sites can be produced byoligonucleotide synthesis and ligation or by site-specific mutagenesistechniques. These analog proteins can be produced in a homogeneous,reduced-carbohydrate form in good yield using yeast expression systems.N-glycosylation sites in eukaryotic proteins are characterized by theamino acids triplet Asn-A₁—Z, where A₁ is any amino acid except Pro, andZ is Ser or Thr. In this sequence, asparagine provides a side chainamino group for covalent attachment of carbohydrate. Such a site can beeliminated by substituting another amino acid for Asn or for residue Z,deleting Asn or Z, or inserting a non-Z amino acid between A₁ and Z, oran amino acid other than Asn between Asn and A₁.

RANK protein derivatives may also be obtained by mutations of the nativeRANK or subunits thereof. A RANK mutated protein, as referred to herein,is a polypeptide homologous to a native RANK protein, respectively, butwhich has an amino acid sequence different from the native proteinbecause of one or a plurality of deletions, insertions or substitutions.The effect of any mutation made in a DNA encoding a mutated peptide maybe easily determined by analyzing the ability of the mutated peptide tobind its counterstructure in a specific manner. Moreover, activity ofRANK analogs, muteins or derivatives can be determined by any of theassays described herein (for example, inhibition of the ability of RANKto activate transcription).

Analogs of the inventive proteins may be constructed by, for example,making various substitutions of residues or sequences or deletingterminal or internal residues or sequences not needed for biologicalactivity. For example, cysteine residues can be deleted or replaced withother amino acids to prevent formation of incorrect intramoleculardisulfide bridges upon renaturation. Other approaches to mutagenesisinvolve modification of adjacent dibasic amino acid residues to enhanceexpression in yeast systems in which KEX2 protease activity is present.

When a deletion or insertion strategy is adopted, the potential effectof the deletion or insertion on biological activity should beconsidered. Subunits of the inventive proteins may be constructed bydeleting terminal or internal residues or sequences. Soluble forms ofRANK can be readily prepared and tested for their ability to inhibitRANK-induced NF-κB activation. Polypeptides corresponding to thecytoplasmic regions, and fragments thereof (for example, a death domain)can be prepared by similar techniques. Additional guidance as to thetypes of mutations that can be made is provided by a comparison of thesequence of RANK to proteins that have similar structures, as well as byperforming structural analysis of the inventive RANK proteins.

Generally, substitutions should be made conservatively; i.e., the mostpreferred substitute amino acids are those which do not affect thebiological activity of RANK (i.e., ability of the inventive proteins tobind antibodies to the corresponding native protein in substantiallyequivalent a manner, the ability to bind the counterstructure insubstantially the same manner as the native protein, the ability totransduce a RANK signal, or ability to induce NF-κB activation uponoverexpression in transient transfection systems, for example). Examplesof conservative substitutions include substitution of amino acidsoutside of the binding domain(s) (either ligand/receptor or antibodybinding areas for the extracellular domain, or regions that interactwith other, intracellular proteins for the cytoplasmic domain), andsubstitution of amino acids that do not alter the secondary and/ortertiary structure of the native protein. Additional examples includesubstituting one aliphatic residue for another, such as Ile, Val, Leu,or Ala for one another, or substitutions of one polar residue foranother, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Othersuch conservative substitutions, for example, substitutions of entireregions having similar hydrophobicity characteristics, are well known.

Mutations in nucleotide sequences constructed for expression of analogproteins or fragments thereof must, of course, preserve the readingframe phase of the coding sequences and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructures such as loops or hairpins which would adversely affecttranslation of the mRNA.

Not all mutations in the nucleotide sequence which encodes a RANKprotein or fragments thereof will be expressed in the final product, forexample, nucleotide substitutions may be made to enhance expression,primarily to avoid secondary structure loops in the transcribed mRNA(see EPA 75,444A, incorporated herein by reference), or to providecodons that are more readily translated by the selected host, e.g., thewell-known E. coli preference codons for E. coli expression.

Although a mutation site may be predetermined, it is not necessary thatthe nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants, randommutagenesis may be conducted and the expressed mutated proteins screenedfor the desired activity. Mutations can be introduced at particular lociby synthesizing oligonucleotides containing a mutant sequence, flankedby restriction sites enabling ligation to fragments of the nativesequence. Following ligation, the resulting reconstructed sequenceencodes an analog having the desired amino acid insertion, substitution,or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

Other embodiments of the inventive proteins include RANK polypeptidesencoded by DNAs capable of hybridizing to the DNA of SEQ ID NO:5 undermoderately stringent conditions (prewashing solution of 5×SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0) and hybridization conditions of 50° C., 5×SSC,overnight) to the DNA sequences encoding RANK, or more preferably understringent conditions (for example, hybridization in 6×SSC at 63° C.overnight; washing in 3×SSC at 55° C.), and other sequences which aredegenerate to those which encode the RANK. In one embodiment, RANKpolypeptides are at least about 70% identical in amino acid sequence tothe amino acid sequence of native RANK protein as set forth in SEQ IDNO:5. In a preferred embodiment, RANK polypeptides are at least about80% identical in amino acid sequence to the native form of RANK; mostpreferred polypeptides are those that are at least about 90% identicalto native RANK.

Percent identity may be determined using a computer program, forexample, the GAP computer program described by Devereux et al. (Nucl.Acids Res. 12:387, 1984) and available from the University of WisconsinGenetics Computer Group (UWGCG). For fragments derived from the RANKprotein, the identity is calculated based on that portion of the RANKprotein that is present in the fragment.

The biological activity of RANK analogs or muteins can be determined bytesting the ability of the analogs or muteins to inhibit activation oftranscription, for example as described in the Examples herein.Alternatively, suitable assays, for example, an enzyme immunoassay or adot blot, employing an antibody that binds native RANK, or a solubleform of RANKL, can be used to assess the activity of RANK analogs ormuteins, as can assays that employ cells expressing RANKL. Suitableassays also include, for example, signal transduction assays and methodsthat evaluate the ability of the cytoplasmic region of RANK to associatewith other intracellular proteins (i.e., TRAFs 2 and 3) involved insignal transduction will also be useful to assess the activity of RANKanalogs or muteins. Such methods are well known in the art.

Fragments of the RANK nucleotide sequences are also useful. In oneembodiment, such fragments comprise at least about 17 consecutivenucleotides, preferably at least about 25 nucleotides, more preferablyat least 30 consecutive nucleotides, of the RANK DNA disclosed herein.DNA and RNA complements of such fragments are provided herein, alongwith both single-stranded and double-stranded forms of the RANK DNA ofSEQ ID NO:5, and those encoding the aforementioned polypeptides. Afragment of RANK DNA generally comprises at least about 17 nucleotides,preferably from about 17 to about 30 nucleotides. Such nucleic acidfragments (for example, a probe corresponding to the extracellulardomain of RANK) are used as a probe or as primers in a polymerase chainreaction (PCR).

The probes also find use in detecting the presence of RANK nucleic acidsin in vitro assays and in such procedures as Northern and Southernblots. Cell types expressing RANK can be identified as well. Suchprocedures are well known, and the skilled artisan can choose a probe ofsuitable length, depending on the particular intended application. ForPCR, 5′ and 3′ primers corresponding to the termini of a desired RANKDNA sequence are employed to amplify that sequence, using conventionaltechniques.

Other useful fragments of the RANK nucleic acids are antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target RANK mRNA (sense) orRANK DNA (antisense) sequences. The ability to create an antisense or asense oligonucleotide, based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988and van der Krol et al., BioTechniques 6:958, 1988.

Uses of DNAs, Proteins and Analogs

The RANK DNAs, proteins, fragments and analogs described herein willhave numerous uses, including the preparation of pharmaceuticalcompositions, as therapeutics in such compositions and as targets foruse in screening assays. For example, soluble forms of RANK will beuseful as antagonists of RANK-mediated NF-κB activation, as well as toinhibit transduction of a signal via RANK. RANK compositions (bothprotein and DNAs) will also be useful in development of both agonisticand antagonistic antibodies to RANK. RANK DNA and DNA encoding RANKfragments are useful in preparing recombinant RANK and RANK fragmentsutilizing host cells transformed with vectors that include the DNA.

The RANK cytoplasmic domain and soluble fragments of the RANKcytoplasmic domain are inhibitors of TRAF/RANK interactions and findutility in in vitro screening assays and in vivo therapeutics. Astherapeutics that are cell membrane permeable, the RANK cytoplasmicdomain and amino acid fragments of the RANK cytoplasmic domain that arenecessary for TRAF binding can be administered to bind one or more TRAFsand antagonize RANK mediated triggering of the NF-κB and/or JNKsignaling pathways. (See examples 20 and 21). Advantageously, solublefragments that specifically disrupt, for example, the TRAF6/RANKassociation or inhibit the TRAF6/RANK association are not likely toeffect signaling from other TNF receptors and are usefulimmunosuppressants or anti-inflammatory agents. Similarly, fragmentsthat enhance or increase TRAF6/RANK association and the resulting TRAF6signaling can be useful immuno upregulators, e.g. provide increased DCfunction. Additionally, a soluble RANK cytoplasmic domain fragment thatspecifically interrupts the TRAF6 mediated RANK signaling that leads toosteoclast differentiation and function can be useful in inhibitingosteoclast formation, and osteoclast activity as well as the effects ofexcessive bone resorption.

Fragments of the RANK cytoplasmic domain useful in pharmaceuticalcompositions include those fragments that are capable of binding TRAFS1, 2, 3, 5, and 6. (See Examples 19 and 20). With respect to TRAF6activity and association, useful RANK cytoplasmic fragments include theCOOH-terminal 72 amino acids (amino acids 545-616 of SEQ ID NO:6), aminoacids 339-422 of SEQ ID NO:6, and amino acids 339-362 of SEQ ID NO:6.RANK fragments that are modulators of RANK association with TRAFS1, 2, 3and 5 include the COOH-terminal 72 amino acids (amino acids 545-616 ofSEQ ID NO:6).

The present invention encompasses methods for screening test compoundsfor their ability to modulate TRAF/RANK interactions and their abilityto modulate activities mediated by TRAF/RANK interactions. Additionally,the present invention includes compounds that modulate RANKF/TRAFinteractions and that are identified by the screening methods of thepresent invention. In general, screening methods involve allowing a RANKor RANK polypeptide fragment that is known to bind or interact with aTRAF, to interact with the TRAF under conditions in which the RANK isknown to bind or interact with the TRAF. The RANK/TRAF interaction isallowed to occur in the presence of a test compound or the test compoundis allowed to contact the RANK/TRAF subsequent to their interaction. Byobserving the effect that the test compound has on the known bindingcharacteristics of the RANK or the RANK fragment, compounds that enhanceRANK/TRAF binding or inhibit RANK/TRAF binding can be identified.

Typical test compounds are small molecules or peptides and may be partof extensive small molecule libraries developed for use in screeningmethods. RANK proteins that may be used in screening methods includefull length RANK, the RANK cytoplasmic domain, RANK fragments, and RANKmutants or RANK analogs that bind a TRAF. Particularly useful RANKfragments include the cytoplasmic domain and the cytoplasmic domainfragments identified above. Particularly, useful TRAFs include TRAF6 andTRAF6/TRAF3, TRAF6/TRAF2 heterocomplexes. TRAFs may be recombinantlyprepared and used directly in cells or isolated. Similarly, native TRAFscan be used in cells or isolated and used in in vitro assays.

Specific screening methods are known in the art and many are extensivelyincorporated in high throughput test systems so that large numbers oftest compounds can be screened within a short amount of time. Suitablescreening methods can be performed in a variety of formats including,but not limited to, binding assay screens, functional assay screens andcell based screens. By observing the affect that test compounds have onTRAF/RANK binding in binding assays, on TRAF/RANK mediated activity infunctional tests and in cell based screens, compounds that are potentialtherapeutics because they can modulate the TRAF/RANK interaction areidentified.

Binding assays and their use in screening methodologies are known in theart. For example, U.S. Pat. No. 5,767,244 (incorporated herein byreference) describes methods useful for screening compounds that areactive at the level of a TRAF6 modulatable cellular function. Inparticular, RANK binding assays can be used to screen for test compoundsthat are capable of modulating TRAF/RANK binding. Suitable assaysinclude standard protein-protein interaction tests that demonstrate thepresence or absence of protein-protein interactions and measure bindingaffinities (See, for example, Example 22). Typically, such bindingassays involve incubating a test mixture under conditions in which thedesired TRAF and RANK protein, RANK fragment or RANK analog binds with aknown binding affinity. Forms of RANK that are particularly useful inscreening for modulators of TRAF6/RANK interaction include the fulllength RANK cytoplasmic domain (amino acids 234-616 of SEQ ID NO:6) andfragments of the RANK cytoplasmic domain that are capable of bindingTRAF6. (See Examples 19 and 20). Such binding fragments include theCOOH-terminal 72 amino acids (amino acids 545-616 of SEQ ID NO:6), aminoacids 339-362 of SEQ ID NO:6, and amino acids 339-422 of SEQ ID NO:6.Forms of RANK that are useful in screening for modulators of RANKassociation with TRAFS 1, 2, 3 and 5 include the COOH-terminal 72 aminoacids (amino acids 545-616 of SEQ ID NO:6). Fragments of theCOOH-terminal 72 amino acids that include amino acids 571-573 are usefulin screening for modulators of RANK association with TRAF3. Similarly,fragments of the COOH-terminal 72 amino acids that include amino acids609-610 are useful in screening for modulators of RANK association withTRAF1, 2 and 5. Modulators that inhibit TRAF/RANK association are usefulTRAF/RANK antagonists and those that enhance TRAF/RANK association areuseful agonists of activities mediated by the TRAF/RANK interaction.

Also The present invention includes polypeptides that bind one or moreTRAFs and have an amino acid sequence that is at least 80% identical toamino acids 545-616, amino acids 339-442, or amino acids 234-616 of SEQID. Identity can be determined using the GAP computer program asdescribed above.

Protein-protein interactions can be observed and measured in bindingassays using a variety of detection methodologies that include, but arenot limited to, surface plasmon resonance (Biacore), radioimmune basedassays, and fluorescence polarization binding assays. When performed inthe presence of a test compound, the ability of the test compound tomodulate (e.g. inhibit or enhance) the protein-protein binding affinityis measured. Test compounds shown to inhibit TRAF/RANK interaction aretherapeutic inhibitors of RANK mediated activities. For example,inhibitors of TRAF6/RANK associations are useful as antagonists of RANKmediated triggering of the NF-κB and/or JNK signaling pathways and canbe immunosupressants or anti-inflammatory therapeutics. TRAF6 bindinginhibitors shown to interrupt the RANK/RANKL signaling process thatleads to osteoclast differentiation are also suitable therapeutics forthe treatment of bone loss and the effects of excess bone resorptionsuch as hypercalcemia. (see Example 2_)

RANK protein, RANK fragments (including RANK cytoplasmic domainfragments identified above), mutants and analogs are also useful in cellbased assay methods that screen for test compounds which are inhibitorsor modulators of the TRAF/RANK interactions. Advantageously, cell basedassays are mechanism based and can be designed to assay test compoundsfor their cell membrane permeability characteristics; their ability tomodulate TRAF/RANK interactions; their ability to selectivity modulate aspecific TRAF/RANK mediated activity; and their cell toxicitycharacteristics. A number of cell based methods are known in the art.Many of the assays are based upon a yeast two-hybrid assay or mammaliantwo-hybrid assay. (See White, Proc. Natl. Acad. Sci. USA 93:10001-10003,1996). Yeast two hybrid assays as they relate to selecting smallmolecule inhibitors of protein-protein interactions are described inHuang et al. Proc. Natl. Acad. Sci 94:13396-13401, 1997. Typically,these assays involve expressing proteins (e.g. RANK and TRAF6) whoseinteraction triggers a reporter gene. Test compounds that are cellpermeable can be identified for their ability to modulate the RANK/TRAF6interaction as noted by a difference in the reporter gene triggering ascompared with the reporter gene triggering in the absence of the testcompound.

Additional assays that are useful for discovering modulators ofRANK/TRAF interactions include in vivo functional assays. For example,test compounds can be screened for their ability to inhibit osteoclastmaturation and/or osteoclast activity by incubating osteoclastprecursors in the presence of CSF-1, RANKL, and a test compound ofinterest under conditions known to induce osteoclast formation or underconditions known to induce osteoclast function. Functional assays thatresult in inhibited or no osteoclast formation or activity can bedetermined by standard TRAP assays as described in Example 23.

DNAs of the present invention are useful for the expression ofrecombinant proteins, and as probes for analysis (either quantitative orqualitative) of the presence or distribution of RANK transcripts.

The inventive proteins will also be useful in preparing kits that areused to detect soluble RANK or RANKL, or monitor RANK-related activity,for example, in patient specimens. RANK proteins will also find uses inmonitoring RANK-related activity in other samples or compositions, as isnecessary when screening for antagonists or mimetics of this activity(for example, peptides or small molecules that inhibit or mimic,respectively, the interaction). A variety of assay formats are useful insuch kits, including (but not limited to) ELISA, dot blot, solid phasebinding assays (such as those using a biosensor), rapid format assaysand bioassays.

The purified RANK according to the invention will facilitate thediscovery of inhibitors of RANK, and thus, inhibitors of an inflammatoryresponse (via inhibition of NF-κB activation). The use of a purifiedRANK polypeptide in the screening for potential inhibitors is importantand can virtually eliminate the possibility of interfering reactionswith contaminants. Such a screening assay can utilize the extracellulardomain of RANK, the intracellular domain, or a fragment of either ofthese polypeptides. Detecting the inhibiting activity of a moleculewould typically involve use of a soluble form of RANK derived from theextracellular domain in a screening assay to detect molecules capable ofbinding RANK and inhibiting binding of, for example, an agonisticantibody or RANKL, or using a polypeptide derived from the intracellulardomain in an assay to detect inhibition of the interaction of RANK andother, intracellular proteins involved in signal transduction.

Moreover, in vitro systems can be used to ascertain the ability ofmolecules to antagonize or agonize RANK activity. Included in suchmethods are uses of RANK chimeras, for example, a chimera of the RANKintracellular domain and an extracellular domain derived from a proteinhaving a known ligand? Utilizing the known ligand to transduce a signalcan then monitor the effects on signal transduction of variousmolecules.

In addition, RANK polypeptides can also be used for structure-baseddesign of RANK-inhibitors. Such structure-based design is also known as“rational drug design.” The RANK polypeptides can be three-dimensionallyanalyzed by, for example, X-ray crystallography, nuclear magneticresonance or homology modeling, all of which are well-known methods. Theuse of RANK structural information in molecular modeling softwaresystems to assist in inhibitor design is also encompassed by theinvention. Such computer-assisted modeling and drug design may utilizeinformation such as chemical conformational analysis, electrostaticpotential of the molecules, protein folding, etc. A particular method ofthe invention comprises analyzing the three dimensional structure ofRANK for likely binding sites of substrates, synthesizing a new moleculethat incorporates a predictive reactive site, and assaying the newmolecule as described above.

Expression of Recombinant RANK

The proteins of the present invention are preferably produced byrecombinant DNA methods by inserting a DNA sequence encoding RANKprotein or an analog thereof into a recombinant expression vector andexpressing the DNA sequence in a recombinant expression system underconditions promoting expression. DNA sequences encoding the proteinsprovided by this invention can be assembled from cDNA fragments andshort oligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene which is capable of being inserted in arecombinant expression vector and expressed in a recombinanttranscriptional unit.

Recombinant expression vectors include synthetic or cDNA-derived DNAfragments encoding RANK, or homologs, muteins or bioequivalent analogsthereof, operably linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. Such regulatory elements include a transcriptional promoter, anoptional operator sequence to control transcription, a sequence encodingsuitable mRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation, as described in detailbelow. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants may additionally be incorporated.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a signal peptide (secretory leader) isoperably linked to DNA for a polypeptide if it is expressed as aprecursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading frame. DNAsequences encoding RANK, or homologs or analogs thereof, which are to beexpressed in a microorganism, will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2:95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (tip) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ P_(L) promoter and cI857ts thermolabile repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl Acad Sci. USA 81:5330, 1984.The leader sequence may be modified to contain, near its 3′ end, one ormore useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the BglI site locatedin the viral origin of replication is included. Further, viral genomicpromoter, control and/or signal sequences may be utilized, provided suchcontrol sequences are compatible with the host cell chosen. Exemplaryvectors can be constructed as disclosed by Okayama and Berg (Mol. Cell.Biol. 3:280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A preferred eukaryotic vector for expression of RANK DNA isreferred to as pDC406 (McMahan et al., EMBO J. 10:2821, 1991), andincludes regulatory sequences derived from SV40, human immunodeficiencyvirus (HIV), and Epstein-Barr virus (EBV). Other preferred vectorsinclude pDC409 and pDC410, which are derived from pDC406. pDC410 wasderived from pDC406 by substituting the EBV origin of replication withsequences encoding the SV40 large T antigen. pDC409 differs from pDC406in that a Bgl II restriction site outside of the multiple cloning sitehas been deleted, making the Bgl II site within the multiple cloningsite unique.

A useful cell line that allows for episomal replication of expressionvectors, such as pDC406 and pDC409, which contain the EBV origin ofreplication, is CV-1/EBNA (ATCC CRL 10478). The CV-1/EBNA cell line wasderived by transfection of the CV-1 cell line with a gene encodingEpstein-Barr virus nuclear antigen-1 (EBNA-1) and constitutively expressEBNA-1 driven from human CMV immediate-early enhancer/promoter.

Host Cells

Transformed host cells are cells which have been transformed ortransfected with expression vectors constructed using recombinant DNAtechniques and which contain sequences encoding the proteins of thepresent invention. Transformed host cells may express the desiredprotein (RANK, or homologs or analogs thereof), but host cellstransformed for purposes of cloning or amplifying the inventive DNA donot need to express the protein. Expressed proteins will preferably besecreted into the culture supernatant, depending on the DNA selected,but may be deposited in the cell membrane.

Suitable host cells for expression of proteins include prokaryotes,yeast or higher eukaryotic cells under the control of appropriatepromoters. Prokaryotes include gram negative or gram positive organisms,for example E. coli or Bacillus spp. Higher eukaryotic cells includeestablished cell lines of mammalian origin as described below. Cell-freetranslation systems could also be employed to produce proteins usingRNAs derived from the DNA constructs disclosed herein. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference.

Prokaryotic expression hosts may be used for expression of RANK, orhomologs or analogs thereof that do not require extensive proteolyticand disulfide processing. Prokaryotic expression vectors generallycomprise one or more phenotypic selectable markers, for example a geneencoding proteins conferring antibiotic resistance or supplying anautotrophic requirement, and an origin of replication recognized by thehost to ensure amplification within the host. Suitable prokaryotic hostsfor transformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

Recombinant RANK may also be expressed in yeast hosts, preferably fromthe Saccharomyces species, such as S. cerevisiae. Yeast of other genera,such as Pichia or Kluyveromyces may also be employed. Yeast vectors willgenerally contain an origin of replication from the 2μ yeast plasmid oran autonomously replicating sequence (ARS), promoter, DNA encoding theprotein, sequences for polyadenylation and transcription termination anda selection gene. Preferably, yeast vectors will include an origin ofreplication and selectable marker permitting transformation of bothyeast and E. coli, e.g., the ampicillin resistance gene of E. coli andS. cerevisiae trp1 gene, which provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, and apromoter derived from a highly expressed yeast gene to inducetranscription of a structural sequence downstream. The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil. Hoststrains transformed by vectors comprising the ADH2 promoter may be grownfor expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect cell culture systems can be employed toexpress recombinant protein. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Examples of suitable mammalian hostcell lines include the COS-7 lines of monkey kidney cells, described byGluzman (Cell 23:175, 1981), and other cell lines capable of expressingan appropriate vector including, for example, CV-1/EBNA (ATCC CRL10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHKcell lines. Mammalian expression vectors may comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences, such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and transcriptional termination sequences.

Purification of Recombinant RANK

Purified RANK, and homologs or analogs thereof are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the DNAs of the present invention, which are then purifiedfrom culture media or cell extracts. For example, supernatants fromsystems which secrete recombinant protein into culture media can befirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit.

Following the concentration step, the concentrate can be applied to asuitable purification matrix. For example, a suitable affinity matrixcan comprise a counter structure protein or lectin or antibody moleculebound to a suitable support. Alternatively, an anion exchange resin canbe employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Gel filtration chromatography also provides a means of purifying theinventive proteins.

Affinity chromatography is a particularly preferred method of purifyingRANK and homologs thereof. For example, a RANK expressed as a fusionprotein comprising an immunoglobulin Fc region can be purified usingProtein A or Protein G affinity chromatography. Moreover, a RANK proteincomprising an oligomerizing zipper domain may be purified on a resincomprising an antibody specific to the oligomerizing zipper domain.Monoclonal antibodies against the RANK protein may also be useful inaffinity chromatography purification, by utilizing methods that arewell-known in the art. A ligand may also be used to prepare an affinitymatrix for affinity purification of RANK.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a RANK composition. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant protein can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Fermentation of yeast which express the inventive protein as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). This reference describes two sequential, reversed-phase HPLCsteps for purification of recombinant human GM-CSF on a preparative HPLCcolumn.

Protein synthesized in recombinant culture is characterized by thepresence of cell components, including proteins, in amounts and of acharacter which depend upon the purification steps taken to recover theinventive protein from the culture. These components ordinarily will beof yeast, prokaryotic or non-human higher eukaryotic origin andpreferably are present in innocuous contaminant quantities, on the orderof less than about 1 percent by weight. Further, recombinant cellculture enables the production of the inventive proteins free of otherproteins which may be normally associated with the proteins as they arefound in nature in the species of origin.

Uses and Administration of RANK Compositions

The present invention provides methods of using therapeutic compositionscomprising an effective amount of a protein and a suitable diluent andcarrier, and methods for regulating an immune or inflammatory response.The use of RANK in conjunction with soluble cytokine receptors orcytokines, or other immunoregulatory molecules is also contemplated.

For therapeutic use, purified protein is administered to a patient,preferably a human, for treatment in a manner appropriate to theindication. Thus, for example, RANK protein compositions administered toregulate immune function can be given by bolus injection, continuousinfusion, sustained release from implants, or other suitable technique.Typically, a therapeutic agent will be administered in the form of acomposition comprising purified RANK, in conjunction withphysiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed.

Ordinarily, the preparation of such protein compositions entailscombining the inventive protein with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrins, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.Preferably, product is formulated as a lyophilizate using appropriateexcipient solutions (e.g., sucrose) as diluents. Appropriate dosages canbe determined in trials. The amount and frequency of administration willdepend, of course, on such factors as the nature and severity of theindication being treated, the desired response, the condition of thepatient, and so forth.

Soluble forms of RANK and other RANK antagonists such as antagonisticmonoclonal antibodies can be administered for the purpose of inhibitingRANK-induced induction of NF-κB activity. NF-κB is a transcriptionfactor that is utilized extensively by cells of the immune system, andplays a role in the inflammatory response. Thus, inhibitors of RANKsignaling will be useful in treating conditions in which signalingthrough RANK has given rise to negative consequences, for example, toxicor septic shock, or graft-versus-host reactions. They may also be usefulin interfering with the role of NF-κB in cellular transformation. Tumorcells are more responsive to radiation when their NF-κB is blocked;thus, soluble RANK (or other antagonists of RANK signaling) will beuseful as an adjunct therapy for disease characterized by neoplasticcells that express RANK.

The following examples are offered by way of illustration, and not byway of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference.

EXAMPLE 1

The example describes the identification and isolation of a DNA encodinga novel member of the TNF receptor superfamily. A partial cDNA insertwith a predicted open reading frame having some similarity to CD40 (acell-surface antigen present on the surface of both normal andneoplastic human B cells that has been shown to play an important rolein B-cell proliferation and differentiation; Stamenkovic et al., EMBO J.8:1403, 1989), was identified in a database containing sequenceinformation from cDNAs generated from human bone marrow-deriveddendritic cells (DC). The insert was excised from the vector byrestriction endonuclease digestion, gel purified. labeled with ³²P, andused to hybridize to colony blots generated from a DC cDNA librarycontaining larger cDNA inserts using high stringency hybridization andwashing techniques (hybridization in 5×SSC, 50% formamide at 42° C.overnight, washing in 0.5×SSC at 63° C.); other suitable high stringencyconditions are disclosed in Sambrook et al. in Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; 1989), 9.52-9.55. Initial experiments yielded a clonereferred to as 9D-8A (SEQ ID NO:1); subsequent analysis indicated thatthis clone contained all but the extreme 5′ end of a novel cDNA, withpredicted intron sequence at the extreme 5′ end (nucleotides 1-92 of SEQID NO:1). Additional colony hybridizations were performed, and a secondclone was isolated. The second clone, referred to as 9D-15C (SEQ IDNO:3), contained the 5′ end without intron interruption but not the full3′end. SEQ ID NO:5 shows the nucleotide and amino acid sequence of apredicted full-length protein based on alignment of the overlappingsequences of SEQ ID NOs:1 and 3.

The encoded protein was designated RANK, for receptor activator ofNF-κB. The cDNA encodes a predicted Type 1 transmembrane protein having616 amino acid residues, with a predicted 24 amino acid signal sequence(the computer predicted cleavage site is after Leu24), a 188 amino acidextracellular domain, a 21 amino acid transmembrane domain, and a 383amino acid cytoplasmic tail. The extracellular region of RANK displayedsignificant amino acid homology (38.5% identity, 52.3% similarity) toCD40. A cloning vector (pBluescriptSK⁻) containing human RANK sequence,designated pBluescript:huRANK (in E. coli DH10B), was deposited with theAmerican Type Culture Collection, Manassas Va. (ATCC) on Dec. 20, 1996,under terms of the Budapest Treaty, and given accession number 98285.

EXAMPLE 2

This example describes construction of a RANK DNA construct to express aRANK/Fc fusion protein. A soluble form of RANK fused to the Fc region ofhuman IgG₁ was constructed in the mammalian expression vector pDC409(U.S. Ser. No. 08/571,579). This expression vector encodes the leadersequence of the Cytomegalovirus (CMV) open reading frame R27080 (SEQ IDNO:9), followed by amino acids 33-213 of RANK, followed by a mutatedform of the constant domain of human IgG₁ that exhibits reduced affinityfor Fc receptors (SEQ ID NO:8; for the fusion protein, the Fc portion ofthe construct consisted of Arg3 through Lys232). An alternativeexpression vector encompassing amino acids 1-213 of RANK (using thenative leader sequence) followed by the IgG₁ mutein was also prepared.Both expression vectors were found to induce high levels of expressionof the RANK/Fc fusion protein in transfected cells.

To obtain RANK/Fc protein, a RANK/Fc expression plasmid is transfectedinto CV-1/EBNA cells, and supernatants are collected for about one week.The RANK/Fc fusion protein is purified by means well-known in the artfor purification of Fc fusion proteins, for example, by protein Asepharose column chromatography according to manufacturer'srecommendations (i.e., Pharmacia, Uppsala, Sweden). SDS-polyacrylamidegel electrophoresis analysis indicted that the purified RANK/Fc proteinmigrated with a molecular weight of ˜55 kDa in the presence of areducing agent, and at a molecular weight of ˜110 kDa in the absence ofa reducing agent.

N-terminal amino acid sequencing of the purified protein made using theCMV R27080 leader showed 60% cleavage after Ala20, 20% cleavage afterPro22 and 20% cleavage after Arg28 (which is the Furin cleavage site;amino acid residues are relative to SEQ ID NO:9); N-terminal amino acidanalysis of the fusion protein expressed with the native leader showedcleavage predominantly after Gln25 (80% after Gln25 and 20% after Arg23;amino acid residues are relative to SEQ ID NO:6, full-length RANK). Bothfusion proteins were able to bind a ligand for RANK is a specific manner(i.e., they bound to the surface of various cell lines such as a murinethymoma cell line, EL4), indicating that the presence of additionalamino acids at the N-terminus of RANK does not interfere with itsability to bind RANKL. Moreover, the construct comprising the CMV leaderencoded RANK beginning at amino acid 33; thus, a RANK peptide having anN-terminus at an amino acid between Arg23 and Pro33, inclusive, isexpected to be able to bind a ligand for RANK in a specific manner.

Other members of the TNF receptor superfamily have a region of aminoacids between the transmembrane domain and the ligand binding domainthat is referred to as a ‘spacer’ region, which is not necessary forligand binding. In RANK, the amino acids between 196 and 213 arepredicted to form such a spacer region. Accordingly, a soluble form ofRANK that terminates with an amino acid in this region is expected toretain the ability to bind a ligand for RANK in a specific manner.Preferred C-terminal amino acids for soluble RANK peptides are selectedfrom the group consisting of amino acids 213 and 196 of SEQ ID NO:6,although other amino acids in the spacer region may be utilized as aC-terminus.

EXAMPLE 3

This example illustrates the preparation of monoclonal antibodiesagainst RANK. Preparations of purified recombinant RANK, for example, ortransfected cells expressing high levels of RANK, are employed togenerate monoclonal antibodies against RANK using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding RANK can also be used as an immunogen, for example, as reviewedby Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies arelikely to be useful in interfering with RANK-induced signaling(antagonistic or blocking antibodies) or in inducing a signal bycross-linking RANK (agonistic antibodies), as components of diagnosticor research assays for RANK or RANK activity, or in affinitypurification of RANK.

To immunize rodents, RANK immunogen is emulsified in an adjuvant (suchas complete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected inamounts ranging from 10-100 μg subcutaneously into a selected rodent,for example, BALB/c mice or Lewis rats. DNA may be given intradermally(Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly(Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has beenfound to be a suitable diluent for DNA-based antigens. Ten days to threeweeks days later, the immunized animals are boosted with additionalimmunogen and periodically boosted thereafter on a weekly, biweekly orevery third week immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith RANK, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004.A preferred screening technique is the antibody capture techniquedescribed by Beckman et al., J. Immunol. 144:4212 (1990). Positiveclones are then injected into the peritoneal cavities of syngeneicrodents to produce ascites containing high concentrations (>1 mg/ml) ofanti-RANK monoclonal antibody. The resulting monoclonal antibody can bepurified by ammonium sulfate precipitation followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to RANK protein.

Monoclonal antibodies were generated using RANK/Fc fusion protein as theimmunogen. These reagents were screened to confirm reactivity againstthe RANK protein. Using the methods described herein to monitor theactivity of the mAbs, both blocking (i.e., antibodies that bind RANK andinhibit binding of a ligand to RANK) and non-blocking (i.e., antibodiesthat bind RANK and do not inhibit ligand binding) were isolated.

EXAMPLE 4

This example illustrates the induction of NF-κB activity by RANK in293/EBNA cells (cell line was derived by transfection of the 293 cellline with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1)that constitutively express EBNA-1 driven from human CMV immediate-earlyenhancer/promoter). Activation of NF-κB activity was measured in293/EBNA cells essentially as described by Yao et al. (Immunity 3:811,1995). Nuclear extracts were prepared and analyzed for NF-κB activity bya gel retardation assay using a 25 base pair oligonucleotide spanningthe NF-κB binding sites. Two million cells were seeded into 10 cm dishestwo days prior to DNA transfection and cultured in DMEM-F12 mediacontaining 2.5% FBS (fetal bovine serum). DNA transfections wereperformed as described herein for the IL-8 promoter/reporter assays.

Nuclear extracts were prepared by solubilization of isolated nuclei with400 mM NaCl (Yao et al., supra). Oligonucleotides containing an NF-κBbinding site were annealed and endlabeled with ³²P using T4 DNApolynucleotide kinase. Mobility shift reactions contained 10 μg ofnuclear extract, 4 μg of poly(dI-dC) and 15,000 cpm labeleddouble-stranded oligonucleotide and incubated at room temperature for 20minutes. Resulting protein-DNA complexes were resolved on a 6% nativepolyacrylamide gel in 0.25×Tris-borate-EDTA buffer.

Overexpression of RANK resulted in induction of NF-κB activity as shownby an appropriate shift in the mobility of the radioactive probe on thegel. Similar results were observed when RANK was triggered by a ligandthat binds RANK and transduces a signal to cells expressing the receptor(i.e., by co-transfecting cells with human RANK a nd murine RANKL DNA;see Example 7 below), and would be expected to occur when triggering isdone with agonistic antibodies.

EXAMPLE 5

This example describes a gene promoter/reporter system based on thehuman Interleukin-8 (IL-8) promoter used to analyze the activation ofgene transcription in vivo. The induction of human IL-8 genetranscription by the cytokines Interleukin-1 (IL-1) or tumor necrosisfactor-alpha (TNF-α) is known to be dependent upon intact NF-κB andNF-IL-6 transcription factor binding sites. Fusion of thecytokine-responsive IL-8 promoter with a cDNA encoding the murine IL-4receptor (mIL-4R) allows measurement of promoter activation by detectionof the heterologous reporter protein (mIL-4R) on the cell surface oftransfected cells.

Human kidney epithelial cells (293/EBNA ) are transfected (via theDEAE/DEXTRAN method) with plasmids encoding 1). the reporter/promoterconstruct (referred to as pIL-8rep), and 2). the cDNA(s) of interest .DNA concentrations are always kept constant by the addition of emptyvector DNA. The 293/EBNA cells are plated at a density of 2.5×10⁴cells/ml (3 ml/well) in a 6 well plate and incubated for two days priorto transfection. Two days after transfection, the mIL-4 receptor isdetected by a radioimmunoassay (RIA) described below.

In one such experiment, the 293/EBNA cells were co-transfected with DNAencoding RANK and with DNA encoding RANKL (see Example 7 below).Co-expression of this receptor and its counterstructure by cells resultsin activation of the signaling process of RANK. For such co-transfectionstudies, the DNA concentration/well for the DEAE transfection were asfollows: 40 ng of pIL-8rep [pBluescriptSK³¹ vector (Stratagene)]; 0.4 ngCD40 (DNA encoding CD40, a control receptor; pCDM8 vector); 0.4 ng RANK(DNA encoding RANK; pDC409 vector), and either 1-50 ng CD40L (DNAencoding the ligand for CD40, which acts as a positive control whenco-transfected with CD40 and as a negative control when co-transfectedwith RANK; in pDC304) or RANKL (DNA encoding a ligand for RANK; inpDC406). Similar experiments can be done using soluble RANKL oragonistic antibodies to RANK to trigger cells transfected with RANK.

For the mIL-4R-specific RIA, a monoclonal antibody reactive with mIL-4Ris labeled with ¹²⁵I via a Chloramine T conjugation method; theresulting specific activity is typically 1.5×10¹⁶ cpm/nmol. After 48hours, transfected cells are washed once with media (DMEM/F12 5% FBS).Non-specific binding sites are blocked by the addition of pre-warmedbinding media containing 5% non-fat dry milk and incubation at 37° C./5%CO₂ in a tissue culture incubator for one hour. The blocking media isdecanted and binding buffer containing ¹²⁵I anti-mIL4R (clone M1; ratIgG1) is added to the cells and incubated with rocking at roomtemperature for 1 hour. After incubation of the cells with theradio-labeled antibody, cells are washed extensively with binding buffer(2×) and twice with phosphate-buffered saline (PBS). Cells are lysed in1 ml of 0.5M NaOH, and total radioactivity is measured with a gammacounter.

Using this assay, 293/EBNA co-transfected with DNAs encoding RANKdemonstrated transcriptional activation, as shown by detection ofmuIL-4R on the cell surface. Overexpression of RANK resulted intranscription of muIL-4R, as did triggering of the RANK by RANKL.Similar results are observed when RANK is triggered by agonisticantibodies.

EXAMPLE 6

This example illustrates the association of RANK with TRAF proteins.Interaction of RANK with cytoplasmic TRAF proteins was demonstrated byco-immunoprecipitation assays essentially as described by Hsu et al.(Cell 84:299; 1996). Briefly, 293/EBNA cells were co-transfected withplasmids that direct the synthesis of RANK and epitope-tagged (FLAG®;SEQ ID NO:7) TRAF2 or TRAF3. Two days after transfection, surfaceproteins were labeled with biotin-ester, and cells were lysed in abuffer containing 0.5% NP-40. RANK and proteins associated with thisreceptor were immunoprecipitated with anti-RANK, washed extensively,resolved by electrophoretic separation on a 6-10% SDS polyacrylamide geland electrophoretically transferred to a nitrocellulose membrane forWestern blotting. The association of TRAF2 and TRAF3 proteins with RANKwas visualized by probing the membrane with an antibody thatspecifically recognizes the FLAG® epitope. TRAFs 2 and 3 did notimmunopreciptitate with anti-RANK in the absence of RANK expression.

EXAMPLE 7

This example describes isolation of a ligand for RANK, referred to asRANKL, by direct expression cloning. The ligand was cloned essentiallyas described in U.S. Ser. No. 08/249,189, filed May 24, 1994 (therelevant disclosure of which is incorporated by reference herein), forCD40L. Briefly, a library was prepared from a clone of a mouse thymomacell line EL-4 (ATCC TIB 39), called EL-40.5, derived by sorting fivetimes with biotinylated CD40/Fc fusion protein in a FACS (fluorescenceactivated cell sorter). The cDNA library was made using standardmethodology; the plasmid DNA was isolated and transfected intosub-confluent CV1-EBNA cells using a DEAE-dextran method. Transfectantswere screened by slide autoradiography for expression of RANKL using atwo-step binding method with RANK/Fc fusion protein as prepared inExample 2 followed by radioiodinated goat anti-human IgG antibody.

A clone encoding a protein that specifically bound RANK was isolated andsequenced; the clone was referred to as 11H. An expression vectorcontaining murine RANKL sequence, designated pDC406:muRANK-L (in E. coliDH10B), was deposited with the American Type Culture Collection,Manassas, Va. (ATCC) on Dec. 20, 1996, under terms of the BudapestTreaty, and given accession number 98284. The nucleotide sequence andpredicted amino acid sequence of this clone are illustrated in SEQ IDNO:10. This clone did not contain an initiator methionine; additional,full-length clones were obtained from a 7B9 library (preparedsubstantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4,1997); the 5′ region was found to be identical to that of human RANKL asshown in SEQ ID NO:12, amino acids 1 through 22, except for substitutionof a Gly for a Thr at residue 9.

This ligand is useful for assessing the ability of RANK to bind RANKL bya number of different assays. For example, transfected cells expressingRANKL can be used in a FACS assay (or similar assay) to evaluate theability of soluble RANK to bind RANKL. Moreover, soluble forms of RANKLcan be prepared and used in assays that are known in the art (i.e.,ELISA or BIAcore assays essentially as described in U.S. Ser. No.08/249,189, filed May 24, 1994). RANKL is also useful in affinitypurification of RANK, and as a reagent in methods to measure the levelsof RANK in a sample. Soluble RANKL is also useful in inducing NF-κBactivation and thus protecting cells that express RANK from apoptosis.

EXAMPLE 8

This example describes the isolation of a human RANK ligand (RANKL)using a PCR-based technique. Murine RANK ligand-specific oligonucleotideprimers were used in PCR reactions using human cell line-derived firststrand cDNAs as templates. Primers corresponded to nucleotides 478497and to the complement of nucleotides 858-878 of murine RANK ligand (SEQID NO:10). An amplified band approximately 400 bp in length from onereaction using the human epidermoid cell line KB (ATCC CCL-17) was gelpurified, and its nucleotide sequence determined; the sequence was 85%identical to the corresponding region of murine RANK ligand, confirmingthat the fragment was from human RANKL.

To obtain full-length human RANKL cDNAs, two human RANKL-specificoligonucleotides derived from the KB PCR product nucleotide sequencewere radiolabeled and used as hybridization probes to screen a human PBLcDNA library prepared in lambda gt10 (Stratagene, La Jolla, Calf.),substantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4,1997. Several positive hybridizing plaques were identified and purified,their inserts subcloned into pBluescript SK⁻ (Stratagene, La Jolla,Calf.), and their nucleotide sequence determined. One isolate, PBL3, wasfound to encode most of the predicted human RANKL, but appeared to bemissing approximately 200 bp of 5′ coding region. A second isolate, PBL5was found to encode much of the predicted human RANKL, including theentire 5′ end and an additional 200 bp of 5′ untranslated sequence.

The 5′ end of PBL5 and the 3′ end of PBL3 were ligated together to forma full length cDNA encoding human RANKL. The nucleotide and predictedamino acid sequence of the full-length human RANK ligand is shown in SEQID NO:12. Human RANK ligand shares 83% nucleotide and 84% amino acididentity with murine RANK ligand. A plasmid vector containing humanRANKL sequence, designated pBluescript:huRANK-L (in E. coli DH10B), wasdeposited with the American Type Culture Collection, Manassas, Va.(ATCC) on Mar. 11, 1997 under terms of the Budapest Treaty, and givenaccession number 98354.

Murine and human RANKL are Type 2 transmembrane proteins. Murine RANKLcontains a predicted 48 amino acid intracellular domain, 21 amino acidtransmembrane domain and 247 amino acid extracellular domain. HumanRANKL contains a predicted 47 amino acid intracellular domain, 21 aminoacid transmembrane domain and 249 amino acid extracellular domain.

EXAMPLE 9

This example describes the chromosomal mapping of human RANK usingPCR-based mapping strategies. Initial human chromosomal assignments weremade using RANK and RANKL-specific PCR primers and a BIOS Somatic CellHybrid PCRable DNA kit from BIOS Laboratories (New Haven, Conn.),following the manufacturer's instructions. RANK mapped to humanchromosome 18; RANK ligand mapped to human chromosome 13. More detailedmapping was performed using a radiation hybrid mapping panel Genebridge4 Radiation Hybrid Panel (Research Genetics, Huntsville, Ala; describedin Walter, MA et al., Nature Genetics 7:22-28, 1994). Data from thisanalysis was then submitted electronically to the MIT Radiation HybridMapper (URL: http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl)following the instructions contained therein. This analysis yieldedspecific genetic marker names which, when submitted electronically tothe NCBI Entrez browser (URL: http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=c&form=0), yielded thespecific map locations. RANK mapped to chromosome 18q22.1, and RANKLmapped to chromosome 13q14.

EXAMPLE 10

This example illustrates the preparation of monoclonal antibodiesagainst RANKL. Preparations of purified recombinant RANKL, for example,or transfixed cells expressing high levels of RANKL, are employed togenerate monoclonal antibodies against RANKL using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding RANKL can also be used as an immunogen, for example, asreviewed by Pardol and Beckerleg in Immunity 3:165, 1995. Suchantibodies are likely to be useful in interfering with RANKL signaling(antagonistic or blocking antibodies), as components of diagnostic orresearch assays for RANKL or RANKL activity, or in affinity purificationof RANKL.

To immunize rodents, RANKL immunogen is emulsified in an adjuvant (suchas complete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected inamounts ranging from 10-100 μg subcutaneously into a selected rodent,for example, BALB/c mice or Lewis rats. DNA may be given intradernally(Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly(Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has beenfound to be a suitable diluent for DNA-based antigens. Ten days to threeweeks days later, the immunized animals are boosted with additionalimmunogen and periodically boosted thereafter on a weekly, biweekly orevery third week immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith RANKL, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004.A preferred screening technique is the antibody capture techniquedescribed by Beckman et al., J. Immunol. 144:4212 (1990). Positiveclones are then injected into the peritoneal cavities of syngeneicrodents to produce ascites containing high concentrations (>1 mg/ml) ofanti-RANK monoclonal antibody. The resulting monoclonal antibody can bepurified by ammonium sulfate precipitation followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to RANKL protein. Using themethods described herein to monitor the activity of the mAbs, bothblocking (i.e., antibodies that bind RANKL and inhibit binding to RANK)and non-blocking (i.e., antibodies that bind RANKL and do not inhibitbinding) are isolated.

EXAMPLE 11

This example demonstrates that RANK expression can be up-regulated.Human peripheral blood T cells were purified by flow cytometry sortingor by negative selection using antibody coated beads, and activated withanti-CD3 (OKT3, Dako) coated plates or phytohemagglutinin in thepresence or absence of various cytokines, including Interleukin-4(IL-4),Transforming Growth Factor-β (TGF-β) and other commerciallyavailable cytokines (IL1-α, IL-2, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12,IL-15, IFN-γ, TNF-α). Expression of RANK was evaluated by FACS in a timecourse experiment for day 2 to day 8, using a mouse monoclonal antibodymAb144 (prepared as described in Example 3), as shown in the tablebelow. Results are expressed as ‘+’ to ‘++++’ referring to the relativeincrease in intensity of staining with anti-RANK. Double labelingexperiments using both anti-RANK and anti-CD8 or anti-CD4 antibodieswere also performed.

TABLE 1 Upregulation of RANK by Cytokines Cytokine (concentration)Results: IL-4 (50 ng/ml) + TGF-β (5 ng/ml) + to ++ IL-4 (50 ng/ml) +TGF-β (5 ng/ml) ++++ IL1-α (10 ng/ml) — IL-2 (20 ng/ml) — IL-3 (25ng/ml) — IL-7 (20 ng/ml) — IL-8 (10 ng/ml) — IL-10 (50 ng/ml) — IL-12(10 ng/ml) —

Of the cytokines tested, IL-4 and TGF-β increased the level of RANKexpression on both CD8+ cytotoxic and CD4+ helper T cells from day 4 today 8. The combination of IL-4 and TGF-β acted synergistically toupregulate expression of this receptor on activated T cells. Thisparticular combination of cytokines is secreted by suppresser T cells,and is believed to be important in the generation of tolerance (reviewedin Mitchison and Sieper, Z. Rheumatol. 54:141, 1995), implicating theinteraction of RANK in regulation of an immune response towards eithertolerance or induction of an active immune response.

EXAMPLE 12

This example illustrates the influence of RANK.Fc and hRANKL onactivated T cell growth. The addition of TGFβ to anti-CD3 activatedhuman peripheral blood T lymphocytes induces proliferation arrest andultimately death of most lymphocytes within the first few days ofculture. We tested the effect of RANK:RANKL interactions on TGFβ-treatedT cells by adding RANK.Fc or soluble human RANKL to T cell cultures.

Human peripheral blood T cells (7×10⁵ PBT) were cultured for six days onanti-CD3 (OKT3, 5 μg/ml) and anti-Flag (M1, 5 μg/ml) coated 24 wellplates in the presence of TGFβ (1 ng/ml) and IL-4 (10 ng/ml), with orwithout recombinant FLAG-tagged soluble hRANKL (1 μg/ml) or RANK.Fc(10μg/ml). Viable T cell recovery was determined by triplicate trypanblue countings.

The addition of RANK.Fc significantly reduced the number of viable Tcells recovered after six days, whereas soluble RANKL greatly increasedthe recovery of viable T cells (FIG. 1). Thus, endogenous or exogenousRANKL enhances the number of viable T cells generated in the presence ofTGFβ. TGFβ, along with IL4, has been implicated in immune responseregulation when secreted by the T_(H)3/regulatory T cell subset. These Tcells are believed to mediate bystander suppression of effector T cells.Accordingly, RANK and its ligand may act in an auto/paracrine fashion toinfluence T cell tolerance. Moreover, TGFβ is known to play a role inthe evasion of the immune system effected by certain pathogenic oropportunistic organisms. In addition to playing a role in thedevelopment of tolerance, RANK may also play a role in immune systemevasion by pathogens.

EXAMPLE 13

This example illustrates the influence of the interaction of RANK onCD1a⁺ dendritic cells (DC). Functionally mature dendritic cells (DC)were generated in vitro from CD34⁺ bone marrow (BM) progenitors.Briefly, human BM cells from normal healthy volunteers were densityfractionated using Ficoll medium and CD34⁺ cells immunoaffinity isolatedusing an anti-CD34 matrix column (Ceprate, CellPro). The CD34⁺ BM cellswere then cultured in human GM-CSF (20 ng/ml), human IL-4 (20 ng/ml),human TNF-α (20 ng/ml), human CHO-derived Flt3L (FL; 100 ng/ml) in SuperMcCoy's medium supplemented with 10% fetal calf serum in a fullyhumidified 37° C. incubator (5% CO2) for 14 days. CD1a⁺, HLA-DR⁺ DC werethen sorted using a FACStar Plus™, and used for biological evaluation ofRANK.

On human CD1a⁺ DC derived from CD34⁺ bone marrow cells, only a subset(20-30%) of CD1a⁺ DC expressed RANK at the cell surface as assessed byflow cytometric analysis. However, addition of CD40L to the DC culturesresulted in RANK surface expression on the majority of CD1a⁺ DC. CD40Lhas been shown to activate DC by enhancing in vitro cluster formation,inducing DC morphological changes and upregulating HLA-DR, CD54, CD58,CD80 and CD86 expression.

Addition of RANKL to DC cultures significantly increased the degree ofDC aggregation and cluster formation above control cultures, similar tothe effects seen with CD40L (FIG. 2). Sorted human CD1a⁺ DC werecultured in a cytokine cocktail (GM-CSF, IL-4, TNF-β and FL) (upper leftpanel), in cocktail plus CD40L (1μg/ml) (upper right), in cocktail plusRANKL (1 μg/ml) (lower left), or in cocktail plus heat inactivated (ΔH)RANKL (1 μg/ml) (lower right) in 24-well flat bottomed culture plates in1 ml culture media for 48-72 hours and then photographed using aninversion microscope. An increase in DC aggregation and clusterformation above control cultures was not evident when heat inactivatedRANKL was used, indicating that this effect was dependent onbiologically active protein. However, initial phenotypic analysis ofadhesion molecule expression indicated that RANKL-induced clustering wasnot due to increased levels of CD2, CD11a, CD54 or CD58.

The addition of RANKL to CD1a⁺ DC enhanced their allo-stimulatorycapacity in a mixed lymphocyte reaction (MLR) by at least 3- to 10-fold,comparable to CD40L-cultured DC (FIG. 3). Allogeneic T cells (1×10⁵)were incubated with varying numbers of irradiated (2000 rad) DC culturedas indicated above for FIG. 2 in 96-well round bottomed culture platesin 0.2 ml culture medium for four days. The cultures were pulsed with0.5 mCi [³H]-thymidine for eight hours and the cells harvested ontoglass fiber sheets for counting on a gas phase β counter. The backgroundcounts for either T cells or DC cultured alone were <100 cpm. Valuesrepresent the mean±SD of triplicate cultures. Heat inactivated RANKL hadno effect. DC allo-stimulatory activity was not further enhanced whenRANKL and CD40L were used in combination, possibly due to DC functionalcapacity having reached a maximal level with either cytokine alone.Neither RANKL nor CD40L enhanced the in vitro growth of DC over thethree day culture period. Unlike CD40L, RANKL did not significantlyincrease the levels of HLA-DR expression nor the expression of CD80 orCD86.

RANKL can enhance DC cluster formation and functional capacity withoutmodulating known molecules involved in cell adhesion (CD18, CD54),antigen presentation (HLA-DR) or costimulation (CD86), all of which areregulated by CD40/CD40L signaling. The lack of an effect on theexpression of these molecules suggests that RANKL may regulate DCfunction via an alternate pathway(s) distinct from CD40/CD40L. Giventhat CD40L regulates RANK surface expression on in vitro-generated DCand that CD40L is upregulated on activated T cells during DC-T cellinteractions, RANK and its ligand may form an important part of theactivation cascade that is induced during DC-mediated T cell expansion.Furthermore, culture of DC in RANKL results in decreased levels ofCD1b/c expression, and increased levels of CD83. Both of these moleculesare similarly modulated during DC maturation by CD40L (Caux et al. J.Exp. Med. 180:1263; 1994), indicating that RANKL induces DC maturation.

Dendritic cells are referred to as “professional” antigen presentingcells, and have a high capacity for sensitizing MHC-restricted T cells.There is growing interest in using dendritic cells ex vivo as tumor orinfectious disease vaccine adjuvants (see, for example, Romani, et al.,J. Exp. Med., 180:83, 1994). Therefore, an agent such as RANKL thatinduces DC maturation and enhances the ability of dendritic cells tostimulate an immune response is likely to be useful in immunotherapy ofvarious diseases.

EXAMPLE 14

This example describes the isolation of the murine homolog of RANK,referred to as muRANK. MuRANK was isolated by a combination ofcross-species PCR and colony hybridization. The conservation of Cysresidues in the Cys-rich pseudorepeats of the extracellular domains ofTNFR superfamily member proteins was exploited to design humanRANK-based PCR primers to be used on murine first strand cDNAs fromvarious sources. Both the sense upstream primer and the antisensedownstream primer were designed to have their 3′ ends terminate withinCys residues.

The upstream sense primer encoded nucleotides 272-295 of SEQ ID NO:5(region encoding amino acids 79-86); the downstream antisense primerencoded the complement of nucleotides 409-427 (region encoding aminoacids 124-130). Standard PCR reactions were set up and run, using theseprimers and first strand cDNAs from various murine cell line or tissuesources. Thirty reaction cycles of 94° C. for 30 seconds, 50° C. for 30seconds, and 72° C. for 20 seconds were run. PCR products were analyzedby electrophoresis, and specific bands were seen in several samples. Theband from one sample was gel purified and DNA sequencing revealed thatthe sequence between the primers was approximately 85% identical to thecorresponding human RANK nucleotide sequence.

A plasmid based cDNA library prepared from the murine fetal liverepithelium line FLE18 (one of the cell lines identified as positive inthe PCR screen) was screened for full-length RANK cDNAs using murineRANK-specific oligonucleotide probes derived from the murine RANKsequence determined from sequencing the PCR product. Two cDNAs, oneencoding the 5′ end and one encoding the 3′ end of full-length murineRANK (based on sequence comparison with the full-length human RANK) wererecombined to generate a full-length murine RANK cDNA. The nucleotideand amino acid sequence of muRANK are shown in SEQ ID Nos:14 and 15.

The cDNA encodes a predicted Type 1 transmembrane protein having 625amino acid residues, with a predicted 30 amino acid signal sequence, a184 amino acid extracellular domain, a 21 amino acid transmembranedomain, and a 390 amino acid cytoplasmic tail. The extracellular regionof muRANK displayed significant amino acid homology (69.7% identity,80.8% similarity) to huRANK. Those of skill in the art will recognizethat the actual cleavage site can be different from that predicted bycomputer; accordingly, the N-terminal of RANK may be from amino acid 25to amino acid 35.

Other members of the TNF receptor superfamily have a region of aminoacids between the transmembrane domain and the ligand binding domainthat is referred to as a ‘spacer’ region, which is not necessary forligand binding. In muRANK, the amino acids between 197 and 214 arepredicted to form such a spacer region. Accordingly, a soluble form ofRANK that terminates with an amino acid in this region is expected toretain the ability to bind a ligand for RANK in a specific manner.Preferred C-terminal amino acids for soluble RANK peptides are selectedfrom the group consisting of amino acids 214, and 197 of SEQ ID NO:14,although other amino acids in the spacer region may be utilized as aC-terminus.

EXAMPLE 15

This example illustrates the preparation of several different solubleforms of RANK and RANKL. Standard techniques of restriction enzymecutting and ligation, in combination with PCR-based isolation offragments for which no convenient restriction sites existed, were used.When PCR was utilized, PCR products were sequenced to ascertain whetherany mutations had been introduced; no such mutations were found.

In addition to the huRANK/Fc described in Example 2, another RANK/Fcfusion protein was prepared by ligating DNA encoding amino acids 1-213of SEQ ID NO:6, to DNA encoding amino acids 3-232 of the Fc muteindescribed previously (SEQ ID NO:8). A similar construct was prepared formurine RANK, ligating DNA encoding amino acids 1-213 of full-lengthmurine RANK (SEQ ID NO:15) to DNA encoding amino acids 3-232 of the Fcmutein (SEQ ID NO:8).

A soluble, tagged, poly-His version of huRANKL was prepared by ligatingDNA encoding the leader peptide from the immunoglobulin kappa chain (SEQID NO:16) to DNA encoding a short version of the FLAG™ tag (SEQ IDNO:17), followed by codons encoding Gly Ser, then a poly-His tag (SEQ IDNO:18), followed by codons encoding Gly Thr Ser, and DNA encoding aminoacids 138-317 of SEQ ID NO:13. A soluble, poly-His tagged version ofmurine RANKL was prepared by ligating DNA encoding the CMV leader (SEQID NO:9) to codons encoding Arg Thr Ser, followed by DNA encodingpoly-His (SEQ ID NO:18) followed by DNA encoding amino acids 119-294 ofSEQ ID NO:11.

A soluble, oligomeric form of huRANKL was prepared by ligating DNAencoding the CMV leader (SEQ ID NO:9) to a codon encoding Asp followedby DNA ending a trimer-former “leucine” zipper (SEQ ID NO:19), then bycodons encoding Thr Arg Ser followed by amino acids 138-317 of SEQ IDNO:13.

These and other constructs are prepared by routine experimentation. Thevarious DNAs are then inserted into a suitable expression vector, andexpressed. Particularly preferred expression vectors are those which canbe used in mammalian cells. For example, pDC409 and pDC304, describedherein, are useful for transient expression. For stable transfection,the use of CHO cells is preferred; several useful vectors are describedin U.S. Ser. No. 08/785,150, now allowed, for example, one of the 2A5-3λ-derived expression vectors discussed therein.

EXAMPLE 16

This example demonstrates that RANKL expression can be up-regulated onmurine T cells. Cells were obtained from mesenteric lymph nodes ofC57BL/6 mice, and activated with anti-CD3 coated plates, Concanavalin A(ConA) or phorbol myristate acetate in combination with ionomycin(anti-CD3: 500A2; Immunex Corporation, Seattle Wash.; ConA, PMA,ionomycin, Sigma, St. Louis, Mo.) substantially as described herein, andcultured from about 2 to 5 days. Expression of RANKL was evaluated in athree color analysis by FACS, using antibodies to the T cell markersCD4, CD8 and CD45RB, and RANK/Fc, prepared as described herein.

RANKL was not expressed on unstimulated murine T cells. T cellsstimulated with either anti-CD3, ConA, or PMA/ionomycin, showeddifferential expression of RANKL: CD4⁺/CD45RB^(Lo) and CD4⁺/CD45RB^(Hi)cells were positive for RANKL, but CD8+ cells were not. RANKL was notobserved on B cells, similar to results observed with human cells.

EXAMPLE 17

This example illustrates the effects of murine RANKL on cellproliferation and activation. Various cells or cell lines representativeof cells that play a role in an immune response (murine spleen, thymusand lymphnode) were evaluated by culturing them under conditionspromoting their viability, in the presence or absence of RANKL. RANKLdid not stimulate any of the tested cells to proliferate. One cell line,a macrophage cell line referred to as RAW 264.7 (ATCC accession numberTIB 71) exhibited some signs of activation.

RAW cells constitutively produce small amounts of TNF-α. Incubation witheither human or murine RANKL enhanced production of TNF-α by these cellsin a dose dependent manner. The results were not due to contamination ofRANKL preparations with endotoxin, since boiling RANKL for 10 minutesabrogated TNF-α production, whereas a similar treatment of purifiedendotoxin (LPS) did not affect the ability of the LPS to stimulate TNF-αproduction. Despite the fact that RANKL activated the macrophage cellline RAW T64.7 for TNF-α production, neither human RANKL nor murineRANKL stimulated nitric oxide production by these cells.

EXAMPLE 18

This example illustrates the effects of murine RANKL on growth anddevelopment of the thymus in fetal mice. Pregnant mice were injectedwith 1 mg of RANK/Fc or vehicle control protein (murine serum albumin;MSA) on days 13, 16 and 19 of gestation. After birth, the neonatescontinued to be injected with RANK/Fc intraperitoneally (IP) on a dailybasis, beginning at a dose of 1 μg, and doubling the dose about everyfour days, for a final dosage of 4 μg. Neonates were taken at days 1, 8and 15 post birth, their thymuses and spleens harvested and examined forsize, cellularity and phenotypic composition.

A slight reduction in thymic size at day 1 was observed in the neonatesborn to the female injected with RANK/Fc; a similar decrease in size wasnot observed in the control neonates. At day 8, thymic size andcellularity were reduced by about 50% in the RANK/Fc-treated animals ascompared to MSA treated mice. Phenotypic analysis demonstrated that therelative proportions of different T cell populations in the thymus werethe same in the RANK/Fc mice as the control mice, indicating that thedecreased cellularity was due to a global depression in the number ofthymic T cells as opposed to a decrease in a specific population(s). TheRANK/Fc-treated neonates were not significantly different from thecontrol neonates at day 15 with respect to either size, cellularity orphenotype of thymic cells. No significant differences were observed inspleen size, cellularity or composition at any of the time pointsevaluated. The difference in cellularity on day 8 and not on day 15 maysuggest that RANK/Fc may assert its effect early in thymic development.

EXAMPLE 19

This example demonstrates that the C-terminal region of the cytoplasmicdomain of RANK is important for binding of several different TRAFproteins. RANK contains at least two recognizable PXQX(X)T motifs thatare likely TRAF docking sites. Accordingly, the importance of variousregions of the cytoplasmic domain of RANK for TRAF binding wasevaluated. A RANK/GST fusion protein was prepared substantially asdescribed in Smith and Johnson, Gene 67:31 (1988), and used in thepreparation of various truncations as described below.

Comparison of the nucleotide sequence of murine and human RANK indicatedthat there were several conserved regions that could be important forTRAF binding. Accordingly, a PCR-based technique was developed tofacilitate preparation of various C-terminal truncations that wouldretain the conserved regions. PCR primers were designed to introduce astop codon and restriction enzyme site at selected points, yielding thetruncations described in Table 1 below. Sequencing confirmed that noundesired mutations had been introduced in the constructs.

Radio-labeled (³⁵S-Met, Cys) TRAF proteins were prepared by in vitrotranslation using a commercially available reticulocyte lysate kitaccording to manufacturer's instructions (Promega). Truncated GST fusionproteins were purified substantially as described in Smith and Johnson(supra). Briefly, E. coli were transfected with an expression vectorencoding a fusion protein, and induced to express the protein. Thebacteria were lysed, insoluble material removed, and the fusion proteinisolated by precipitation with glutathione-coated beads (Sepahrose 4B,Pharmacia, Uppsala Sweden).

The beads were washed, and incubated with various radiolabeled TRAFproteins. After incubation and wash steps, the fusion protein/TRAFcomplexes were removed from the beads by boiling in 0.1%SDS+β-mercaptoethanol, and loaded onto 12% SDS gels (Novex). The gelswere subjected to autoradiography, and the presence or absence ofradiolabeled material recorded. The results are shown in Table 2 below.

TABLE 2 Binding of Various TRAF Proteins to the Cytoplasmic Domain ofRANK C terminal E206- E206- Truncations: E206-S339 E206-Y421 M476 G544Full length TRAF1 — — — — ++ TRAF2 — — — — ++ TRAF3 — — — — ++ TRAF4 — —— — — TRAF5 — — — — + TRAF6 — + + + ++

These results indicate that TRAF1, TRAF2, TRAF3, TRAF 5 and TRAF6 bindto the most distal portion of the RANK cytoplasmic domain (betweenamino-acid G544 and A616). TRAF6 also has a binding site between S339and Y421. In this experiment, TRAF5 also bound the cytoplasmic domain ofRANK.

In another experiment using the same methods to prepare the RANKcytoplasmic domain fragment and test for TRAF6 binding, TRAF6 bound tothe huRANK cytoplasmic domain fragment amino acids 339-362.

To confirm that the in vitro interaction of TRAFs with RANK also occurin cells, co-immunoprecipitation experiments were performed in 293 cellscotransfected with RANK, or RANK fragments, and tagged TRAFs. Theresults demonstrated that full length RANK associates with TRAF 1, 2, 3,5 and 6. 293 cells containing the RANK G544 deletion (RANK lacking theC-terminal 72 amino acids) coprecipitated with TRAFS 2, 3 and 6. Sincethe in vitro binding experiments showed did not show TRAF2 and TRAF3association with this RANK fragment, these results suggest thatTRAF2/TRAF6 and TRAP3/TRAF6 form heterocomplexes. These results alsosupport the conclusions reached in the experiment described in Example6.

EXAMPLE 20

The following describes the functional effect of RANK cytoplasmicdeletion mutants expressed in 293 cells using NF-κB reporter assays.

A NF-κB-responsive reporter plasmid was constructed in pGL2-Basicobtained from Promega with the human IL-8 promoter containing a NF-κBbinding site fused to a luciferase reporter. RANK cytoplasmic deletionswere subcloned into a full length RANK expression vector pDC304. 293cells from Invitrogen, San Diego, Calf. were transiently transfected bythe DEAE-Dextran method with the reporter plasmid either alone or incombination with the RANK cDNA in pDC304 and reporter activity wasmeasured in 293 cells.

Transfection of 293 cells with the RANK deletion construct lacking theCOOH-terminal 72 amino acids (RANKΔ544) resulted in reduced NF-κBreporter activity in the absence of RANKL activation. This contrastswith RANKL treatment of RANKΔ544-expressing cells which induced NF-κBactivation to levels similar to that seen with full-length RANK. Cellstransfected with RANK constructs having further deletion ofCOOH-terminal sequences had minimal effects on the constitutive andRANKL-mediated reporter activity until the removal of amino acids339-422 (RANKΔ339) which completely abrogated both constitutivesignaling and responsiveness to RANKL. These data indicate that RANKcontains two domains (amino acids 339-422 and amino acids 544-616 of SEQID NO:6) within the cytoplasmic region that are important for NF-κBsignaling. These two regions correspond with the two domains whichaffect binding TRAFS1, 2, 3, 5, and 6 (the domain characterized as aminoacids 544-616 of SEQ ID NO:6) and TRAF6 (the domain characterized asamino acids 339-422 of SEQ ID NO:6). These results also suggest thatRANK's ability to directly bind TRAF6, in the absence of direct bindingof other TRAFs, allows optimal RANK signaling. This is supported by theresults obtained in Example 21 in which signaling through amino acids339-442 results in partially inhibited JNK activity.

EXAMPLE 21

The following describes the functional effect of RANK cytoplasmicdeletion mutants expressed in 293 cells using JNK activation assays. Forthe JNK assays, whole cell extracts were prepared from transfected 293cells 24 hr after transfection. The cells were lysed in a buffer andclarified lysates were immunoprecipitated with anti-JNK (FL) andanti-JNK (C17). The complexes were washed and JNK activity wasdetermined by an immune-complex assay using GST-cJun and [³²]-ATP assubstrates. Reaction products were resolved on SDS/PAGE and visualizedby autoradiography.

Transfection of full length RANK significantly induced JNK activity.Transfection of RANK having a truncated COOH-terminal 72 amino acidsabrogated the majority of JNK activity. Residual JNK activity wascompletely inhibited in cells transfected with RANK having furthertruncation of amino acids 339-422 (SEQ ID NO:6) in the RANK cytoplasmicdomain.

These results demonstrate that two distinct RANK cytoplasmic domains(residues 544-616 and 339-422 of SEQ ID NO:6) play functional roles inJNK activation similar to the domains necessary for NF-κB activationdescribed in Example 20.

EXAMPLE 22

This example describes a plate binding assay useful in comparing theability of various ligands or test compounds to bind receptors. Theassay is performed essentially as described in Smith et al., Virology236:316 (1997). Briefly, 96-well microtiter plates are coated with anantibody to human Fc (i.e., polyclonal goat anti human Fc). Receptor/Fcfusion proteins are then added, and after incubation, the plates arewashed. Serial dilutions of the ligands are then added. The ligands maybe directly labeled (i.e., with ¹²⁵I), or a detecting reagent that isradioactively labeled may be used. After incubation, the plates arewashed, specifically bound ligands are released, and the amount ofligand bound quantified.

Using this method, RANK/Fc and OPG/Fc were bound to 96-well plates. Inan indirect method, a RANKL/zipper fusion is detected using a labeledantibody to the zipper moiety. It was found that human OPG/Fc bindsmRANKL at 0.05 nM, and human RANK/Fc binds mRANKL at 0.1 nM. Thesevalues indicate similar binding affinities of OPG and RANK for RANKL,confirming the utility of RANK as an inhibitor of osteoclast activity ina manner similar to OPG.

EXAMPLE 23

The following describes the formation of osteoclast like cells from bonemarrow cell cultures using a soluble RANKL in the form of solubleRANKL/leucine zipper fusion protein (RANKL LZ).

Using RANKL LZ at 1 μg/ml, osteoclasts were generated from murine bonemarrow (BM) in the presence of CSF-1. These osteoclasts are formed bythe fusion of macrophage-like cells and are characterized by their TRAP(tartrate-resistant acid phosphatase) positivity. No TRAP⁺ cells wereseen in cultures containing CSF-1 alone or in cultures containing CSF-1and TRAIL LZ (a control for the soluble RANKL LZ). Even though human andmonkey bone marrow contains more contaminating fibroblasts than murinebone marrow, osteoclasts were generated from murine and monkey bonemarrow with the combination of CSF-1 and soluble RANKL LZ. In adose-response study using murine bone marrow and suboptimal amounts ofCSF-1 (40 ng/ml), the effects of soluble RANKL LZ plateaued at about 100ng/ml.

The effect of soluble RANKL LZ on proliferation of cells was studied inthe same cultures using Alamar Blue. After 5 days, the proliferativeresponse was lower in cultures containing CSF-1 and RANKL LZ than inthose containing CSF-1 alone. The supports the observation that solubleRANKL LZ is inducing osteoclast differentiation. When CSF-1 and RANKL LZare washed out of murine BM cultures at day 7 or 8, cells do not surviveif they are recultured in medium or in RANKL LZ alone. In contrast,cells do survive if recultured in CSF-1. When RANKL LZ was added tothese cultures there was no added benefit. Thus, the combination ofCSF-1 and RANKL are required for the generation of osteoclast.Additionally, once formed, CSF-1 is sufficient to maintain theirsurvival in culture.

Finally, using human bone marrow, soluble anti-human RANK mAb andimmobilized anti-human RANK mAb were compared to RANKL LZ for thegeneration of osteoclasts in the presence of CSF-1. Immobilized M331 andRANKL LZ were found to be equally effective for osteoclast generationwhile soluble M331 was superior to both immobilized antibody and RANKLLZ. This confirms that the osteoclast differentiating activity of RANKLis mediated through RANK rather than via an alternative receptor.

Since osteoclasts cannot readily be harvested and analyzed by flowcytometry, ¹²⁵I-labeled calcitonin binding assays were used to identifyosteoclasts (the calcitonin receptor is considered to be anosteoclast-specific marker). Osteoclasts generated from murine BMcultured with CSF-1and RANKL LZ for 9 days showed binding ofradiolabeled calcitonin confirming their osteoclast identity.

The above confirms that RANK/RANKL binding is important in myeloid celland osteoclast differentiation. The results described in Example 2_suggest that if the TRAF6 binding domain is absent in RANK, NF-κKtranscription factor activity induced by RANK/RANKL interaction isnon-existent. Since it is known that mice lacking two NF-κB subunitshave osteoclast defects, there is strong suggestion that TRAF6 isespecially important in mediating RANK signals in the osteoclastdifferentiation pathway.

EXAMPLE 24

In order to determine RANKL expression by either of two differentsquamous cell carcinomas, standard Western blot and RT-PCR studies wereperformed on MH-85 and OKK cells. One of these carcinoma cells, theMH-85 cells, is associated with hypercalcemia.

The results confirmed that MH-85 and OKK squamous cells express RANKL.MH-85 cells, in addition to being linked with hypercalcemia in patientsinflicted with this carcinoma, also express M-CSF (CSF-1). It was alsodetermined that CSF-1 upregulates RANK expression on osteoclastprecursors. The enhanced amount of CSF-1 in MH-85 type squamous cellcancer patients can lead to an upregulation of RANK and increased RANKinteraction with RANKL. Signals transduced by RANK and RANKL interactionresult in increased numbers of mature osteoclasts and bone breakdown.Since soluble forms of RANK can inhibit the RANK/RANKL interaction,administering a soluble form of RANK (e.g. the extracellular region ofRANK fused to an Fc) to a squamous cell cancer patient provides relieffrom adverse effects of this cancer, including hypercalcernia.

19 3115 base pairs nucleic acid single linear cDNA NO NO HOMO SAPIENSBONE-MARROW DERIVED DENDRITIC CELLS 9D-8A CDS 93..1868 1 GCTGCTGCTGCTCTGCGCGC TGCTCGCCCG GCTGCAGTTT TATCCAGAAA GAGCTGTGTG 60 GACTCTCTGCCTGACCTCAG TGTTCTTTTC AG GTG GCT TTG CAG ATC GCT CCT 113 Val Ala Leu GlnIle Ala Pro 1 5 CCA TGT ACC AGT GAG AAG CAT TAT GAG CAT CTG GGA CGG TGCTGT AAC 161 Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg Cys CysAsn 10 15 20 AAA TGT GAA CCA GGA AAG TAC ATG TCT TCT AAA TGC ACT ACT ACCTCT 209 Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser25 30 35 GAC AGT GTA TGT CTG CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG257 Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 4045 50 55 AAT GAA GAA GAT AAA TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG305 Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys 6065 70 GCC CTG GTG GCC GTG GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC353 Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys 7580 85 GCG TGC ACG GCT GGG TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC401 Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 9095 100 CGC AAC ACC GAG TGC GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG449 Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln 105110 115 CTC AAC AAG GAC ACA GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT497 Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 120125 130 135 GAT GCC TTT TCC TCC ACG GAC AAA TGC AGA CCC TGG ACC AAC TGTACC 545 Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr140 145 150 TTC CTT GGA AAG AGA GTA GAA CAT CAT GGG ACA GAG AAA TCC GATGCG 593 Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala155 160 165 GTT TGC AGT TCT TCT CTG CCA GCT AGA AAA CCA CCA AAT GAA CCCCAT 641 Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His170 175 180 GTT TAC TTG CCC GGT TTA ATA ATT CTG CTT CTC TTC GCG TCT GTGGCC 689 Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala185 190 195 CTG GTG GCT GCC ATC ATC TTT GGC GTT TGC TAT AGG AAA AAA GGGAAA 737 Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys200 205 210 215 GCA CTC ACA GCT AAT TTG TGG CAC TGG ATC AAT GAG GCT TGTGGC CGC 785 Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys GlyArg 220 225 230 CTA AGT GGA GAT AAG GAG TCC TCA GGT GAC AGT TGT GTC AGTACA CAC 833 Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser ThrHis 235 240 245 ACG GCA AAC TTT GGT CAG CAG GGA GCA TGT GAA GGT GTC TTACTG CTG 881 Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly Val Leu LeuLeu 250 255 260 ACT CTG GAG GAG AAG ACA TTT CCA GAA GAT ATG TGC TAC CCAGAT CAA 929 Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp Met Cys Tyr Pro AspGln 265 270 275 GGT GGT GTC TGT CAG GGC ACG TGT GTA GGA GGT GGT CCC TACGCA CAA 977 Gly Gly Val Cys Gln Gly Thr Cys Val Gly Gly Gly Pro Tyr AlaGln 280 285 290 295 GGC GAA GAT GCC AGG ATG CTC TCA TTG GTC AGC AAG ACCGAG ATA GAG 1025 Gly Glu Asp Ala Arg Met Leu Ser Leu Val Ser Lys Thr GluIle Glu 300 305 310 GAA GAC AGC TTC AGA CAG ATG CCC ACA GAA GAT GAA TACATG GAC AGG 1073 Glu Asp Ser Phe Arg Gln Met Pro Thr Glu Asp Glu Tyr MetAsp Arg 315 320 325 CCC TCC CAG CCC ACA GAC CAG TTA CTG TTC CTC ACT GAGCCT GGA AGC 1121 Pro Ser Gln Pro Thr Asp Gln Leu Leu Phe Leu Thr Glu ProGly Ser 330 335 340 AAA TCC ACA CCT CCT TTC TCT GAA CCC CTG GAG GTG GGGGAG AAT GAC 1169 Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu Glu Val Gly GluAsn Asp 345 350 355 AGT TTA AGC CAG TGC TTC ACG GGG ACA CAG AGC ACA GTGGGT TCA GAA 1217 Ser Leu Ser Gln Cys Phe Thr Gly Thr Gln Ser Thr Val GlySer Glu 360 365 370 375 AGC TGC AAC TGC ACT GAG CCC CTG TGC AGG ACT GATTGG ACT CCC ATG 1265 Ser Cys Asn Cys Thr Glu Pro Leu Cys Arg Thr Asp TrpThr Pro Met 380 385 390 TCC TCT GAA AAC TAC TTG CAA AAA GAG GTG GAC AGTGGC CAT TGC CCG 1313 Ser Ser Glu Asn Tyr Leu Gln Lys Glu Val Asp Ser GlyHis Cys Pro 395 400 405 CAC TGG GCA GCC AGC CCC AGC CCC AAC TGG GCA GATGTC TGC ACA GGC 1361 His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp ValCys Thr Gly 410 415 420 TGC CGG AAC CCT CCT GGG GAG GAC TGT GAA CCC CTCGTG GGT TCC CCA 1409 Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu ValGly Ser Pro 425 430 435 AAA CGT GGA CCC TTG CCC CAG TGC GCC TAT GGC ATGGGC CTT CCC CCT 1457 Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly Met GlyLeu Pro Pro 440 445 450 455 GAA GAA GAA GCC AGC AGG ACG GAG GCC AGA GACCAG CCC GAG GAT GGG 1505 Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp GlnPro Glu Asp Gly 460 465 470 GCT GAT GGG AGG CTC CCA AGC TCA GCG AGG GCAGGT GCC GGG TCT GGA 1553 Ala Asp Gly Arg Leu Pro Ser Ser Ala Arg Ala GlyAla Gly Ser Gly 475 480 485 AGC TCC CCT GGT GGC CAG TCC CCT GCA TCT GGAAAT GTG ACT GGA AAC 1601 Ser Ser Pro Gly Gly Gln Ser Pro Ala Ser Gly AsnVal Thr Gly Asn 490 495 500 AGT AAC TCC ACG TTC ATC TCC AGC GGG CAG GTGATG AAC TTC AAG GGC 1649 Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val MetAsn Phe Lys Gly 505 510 515 GAC ATC ATC GTG GTC TAC GTC AGC CAG ACC TCGCAG GAG GGC GCG GCG 1697 Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser GlnGlu Gly Ala Ala 520 525 530 535 GCG GCT GCG GAG CCC ATG GGC CGC CCG GTGCAG GAG GAG ACC CTG GCG 1745 Ala Ala Ala Glu Pro Met Gly Arg Pro Val GlnGlu Glu Thr Leu Ala 540 545 550 CGC CGA GAC TCC TTC GCG GGG AAC GGC CCGCGC TTC CCG GAC CCG TGC 1793 Arg Arg Asp Ser Phe Ala Gly Asn Gly Pro ArgPhe Pro Asp Pro Cys 555 560 565 GGC GGC CCC GAG GGG CTG CGG GAG CCG GAGAAG GCC TCG AGG CCG GTG 1841 Gly Gly Pro Glu Gly Leu Arg Glu Pro Glu LysAla Ser Arg Pro Val 570 575 580 CAG GAG CAA GGC GGG GCC AAG GCT TGAGCGCCCCCCA TGGCTGGGAG 1888 Gln Glu Gln Gly Gly Ala Lys Ala 585 590CCCGAAGCTC GGAGCCAGGG CTCGCGAGGG CAGCACCGCA GCCTCTGCCC CAGCCCCGGC 1948CACCCAGGGA TCGATCGGTA CAGTCGAGGA AGACCACCCG GCATTCTCTG CCCACTTTGC 2008CTTCCAGGAA ATGGGCTTTT CAGGAAGTGA ATTGATGAGG ACTGTCCCCA TGCCCACGGA 2068TGCTCAGCAG CCCGCCGCAC TGGGGCAGAT GTCTCCCCTG CCACTCCTCA AACTCGCAGC 2128AGTAATTTGT GGCACTATGA CAGCTATTTT TATGACTATC CTGTTCTGTG GGGGGGGGGT 2188CTATGTTTTC CCCCCATATT TGTATTCCTT TTCATAACTT TTCTTGATAT CTTTCCTCCC 2248TCTTTTTTAA TGTAAAGGTT TTCTCAAAAA TTCTCCTAAA GGTGAGGGTC TCTTTCTTTT 2308CTCTTTTCCT TTTTTTTTTC TTTTTTTGGC AACCTGGCTC TGGCCCAGGC TAGAGTGCAG 2368TGGTGCGATT ATAGCCCGGT GCAGCCTCTA ACTCCTGGGC TCAAGCAATC CAAGTGATCC 2428TCCCACCTCA ACCTTCGGAG TAGCTGGGAT CACAGCTGCA GGCCACGCCC AGCTTCCTCC 2488CCCCGACTCC CCCCCCCCAG AGACACGGTC CCACCATGTT ACCCAGCCTG GTCTCAAACT 2548CCCCAGCTAA AGCAGTCCTC CAGCCTCGGC CTCCCAAAGT ACTGGGATTA CAGGCGTGAG 2608CCCCCACGCT GGCCTGCTTT ACGTATTTTC TTTTGTGCCC CTGCTCACAG TGTTTTAGAG 2668ATGGCTTTCC CAGTGTGTGT TCATTGTAAA CACTTTTGGG AAAGGGCTAA ACATGTGAGG 2728CCTGGAGATA GTTGCTAAGT TGCTAGGAAC ATGTGGTGGG ACTTTCATAT TCTGAAAAAT 2788GTTCTATATT CTCATTTTTC TAAAAGAAAG AAAAAAGGAA ACCCGATTTA TTTCTCCTGA 2848ATCTTTTTAA GTTTGTGTCG TTCCTTAAGC AGAACTAAGC TCAGTATGTG ACCTTACCCG 2908CTAGGTGGTT AATTTATCCA TGCTGGCAGA GGCACTCAGG TACTTGGTAA GCAAATTTCT 2968AAAACTCCAA GTTGCTGCAG CTTGGCATTC TTCTTATTCT AGAGGTCTCT CTGGAAAAGA 3028TGGAGAAAAT GAACAGGACA TGGGGCTCCT GGAAAGAAAG GGCCCGGGAA GTTCAAGGAA 3088GAATAAAGTT GAAATTTTAA AAAAAAA 3115 591 amino acids amino acid linearprotein not provided 2 Val Ala Leu Gln Ile Ala Pro Pro Cys Thr Ser GluLys His Tyr Glu 1 5 10 15 His Leu Gly Arg Cys Cys Asn Lys Cys Glu ProGly Lys Tyr Met Ser 20 25 30 Ser Lys Cys Thr Thr Thr Ser Asp Ser Val CysLeu Pro Cys Gly Pro 35 40 45 Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu AspLys Cys Leu Leu His 50 55 60 Lys Val Cys Asp Thr Gly Lys Ala Leu Val AlaVal Val Ala Gly Asn 65 70 75 80 Ser Thr Thr Pro Arg Arg Cys Ala Cys ThrAla Gly Tyr His Trp Ser 85 90 95 Gln Asp Cys Glu Cys Cys Arg Arg Asn ThrGlu Cys Ala Pro Gly Leu 100 105 110 Gly Ala Gln His Pro Leu Gln Leu AsnLys Asp Thr Val Cys Lys Pro 115 120 125 Cys Leu Ala Gly Tyr Phe Ser AspAla Phe Ser Ser Thr Asp Lys Cys 130 135 140 Arg Pro Trp Thr Asn Cys ThrPhe Leu Gly Lys Arg Val Glu His His 145 150 155 160 Gly Thr Glu Lys SerAsp Ala Val Cys Ser Ser Ser Leu Pro Ala Arg 165 170 175 Lys Pro Pro AsnGlu Pro His Val Tyr Leu Pro Gly Leu Ile Ile Leu 180 185 190 Leu Leu PheAla Ser Val Ala Leu Val Ala Ala Ile Ile Phe Gly Val 195 200 205 Cys TyrArg Lys Lys Gly Lys Ala Leu Thr Ala Asn Leu Trp His Trp 210 215 220 IleAsn Glu Ala Cys Gly Arg Leu Ser Gly Asp Lys Glu Ser Ser Gly 225 230 235240 Asp Ser Cys Val Ser Thr His Thr Ala Asn Phe Gly Gln Gln Gly Ala 245250 255 Cys Glu Gly Val Leu Leu Leu Thr Leu Glu Glu Lys Thr Phe Pro Glu260 265 270 Asp Met Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln Gly Thr CysVal 275 280 285 Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg Met LeuSer Leu 290 295 300 Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg GlnMet Pro Thr 305 310 315 320 Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln ProThr Asp Gln Leu Leu 325 330 335 Phe Leu Thr Glu Pro Gly Ser Lys Ser ThrPro Pro Phe Ser Glu Pro 340 345 350 Leu Glu Val Gly Glu Asn Asp Ser LeuSer Gln Cys Phe Thr Gly Thr 355 360 365 Gln Ser Thr Val Gly Ser Glu SerCys Asn Cys Thr Glu Pro Leu Cys 370 375 380 Arg Thr Asp Trp Thr Pro MetSer Ser Glu Asn Tyr Leu Gln Lys Glu 385 390 395 400 Val Asp Ser Gly HisCys Pro His Trp Ala Ala Ser Pro Ser Pro Asn 405 410 415 Trp Ala Asp ValCys Thr Gly Cys Arg Asn Pro Pro Gly Glu Asp Cys 420 425 430 Glu Pro LeuVal Gly Ser Pro Lys Arg Gly Pro Leu Pro Gln Cys Ala 435 440 445 Tyr GlyMet Gly Leu Pro Pro Glu Glu Glu Ala Ser Arg Thr Glu Ala 450 455 460 ArgAsp Gln Pro Glu Asp Gly Ala Asp Gly Arg Leu Pro Ser Ser Ala 465 470 475480 Arg Ala Gly Ala Gly Ser Gly Ser Ser Pro Gly Gly Gln Ser Pro Ala 485490 495 Ser Gly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly500 505 510 Gln Val Met Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val SerGln 515 520 525 Thr Ser Gln Glu Gly Ala Ala Ala Ala Ala Glu Pro Met GlyArg Pro 530 535 540 Val Gln Glu Glu Thr Leu Ala Arg Arg Asp Ser Phe AlaGly Asn Gly 545 550 555 560 Pro Arg Phe Pro Asp Pro Cys Gly Gly Pro GluGly Leu Arg Glu Pro 565 570 575 Glu Lys Ala Ser Arg Pro Val Gln Glu GlnGly Gly Ala Lys Ala 580 585 590 1391 base pairs nucleic acid singlelinear cDNA NO NO HOMO SAPIENS BONE-MARROW DERIVED DENDRITIC CELLS9D-15C CDS 39..1391 3 CCGCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCGCC ATG GCCCCG CGC GCC 53 Met Ala Pro Arg Ala 1 5 CGG CGG CGC CGC CCG CTG TTC GCGCTG CTG CTG CTC TGC GCG CTG CTC 101 Arg Arg Arg Arg Pro Leu Phe Ala LeuLeu Leu Leu Cys Ala Leu Leu 10 15 20 GCC CGG CTG CAG GTG GCT TTG CAG ATCGCT CCT CCA TGT ACC AGT GAG 149 Ala Arg Leu Gln Val Ala Leu Gln Ile AlaPro Pro Cys Thr Ser Glu 25 30 35 AAG CAT TAT GAG CAT CTG GGA CGG TGC TGTAAC AAA TGT GAA CCA GGA 197 Lys His Tyr Glu His Leu Gly Arg Cys Cys AsnLys Cys Glu Pro Gly 40 45 50 AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCTGAC AGT GTA TGT CTG 245 Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser AspSer Val Cys Leu 55 60 65 CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AATGAA GAA GAT AAA 293 Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn GluGlu Asp Lys 70 75 80 85 TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCCCTG GTG GCC GTG 341 Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala LeuVal Ala Val 90 95 100 GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCGTGC ACG GCT GGG 389 Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala CysThr Ala Gly 105 110 115 TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGCAAC ACC GAG TGC 437 Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg AsnThr Glu Cys 120 125 130 GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTCAAC AAG GAC ACA 485 Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln Leu AsnLys Asp Thr 135 140 145 GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GATGCC TTT TCC TCC 533 Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp AlaPhe Ser Ser 150 155 160 165 ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACCTTC CTT GGA AAG AGA 581 Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr PheLeu Gly Lys Arg 170 175 180 GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCGGTT TGC AGT TCT TCT 629 Val Glu His His Gly Thr Glu Lys Ser Asp Ala ValCys Ser Ser Ser 185 190 195 CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CATGTT TAC TTG CCC GGT 677 Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His ValTyr Leu Pro Gly 200 205 210 TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCCCTG GTG GCT GCC ATC 725 Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala LeuVal Ala Ala Ile 215 220 225 ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAAGCA CTC ACA GCT AAT 773 Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys AlaLeu Thr Ala Asn 230 235 240 245 TTG TGG CAC TGG ATC AAT GAG GCT TGT GGCCGC CTA AGT GGA GAT AAG 821 Leu Trp His Trp Ile Asn Glu Ala Cys Gly ArgLeu Ser Gly Asp Lys 250 255 260 GAG TCC TCA GGT GAC AGT TGT GTC AGT ACACAC ACG GCA AAC TTT GGT 869 Glu Ser Ser Gly Asp Ser Cys Val Ser Thr HisThr Ala Asn Phe Gly 265 270 275 CAG CAG GGA GCA TGT GAA GGT GTC TTA CTGCTG ACT CTG GAG GAG AAG 917 Gln Gln Gly Ala Cys Glu Gly Val Leu Leu LeuThr Leu Glu Glu Lys 280 285 290 ACA TTT CCA GAA GAT ATG TGC TAC CCA GATCAA GGT GGT GTC TGT CAG 965 Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp GlnGly Gly Val Cys Gln 295 300 305 GGC ACG TGT GTA GGA GGT GGT CCC TAC GCACAA GGC GAA GAT GCC AGG 1013 Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala GlnGly Glu Asp Ala Arg 310 315 320 325 ATG CTC TCA TTG GTC AGC AAG ACC GAGATA GAG GAA GAC AGC TTC AGA 1061 Met Leu Ser Leu Val Ser Lys Thr Glu IleGlu Glu Asp Ser Phe Arg 330 335 340 CAG ATG CCC ACA GAA GAT GAA TAC ATGGAC AGG CCC TCC CAG CCC ACA 1109 Gln Met Pro Thr Glu Asp Glu Tyr Met AspArg Pro Ser Gln Pro Thr 345 350 355 GAC CAG TTA CTG TTC CTC ACT GAG CCTGGA AGC AAA TCC ACA CCT CCT 1157 Asp Gln Leu Leu Phe Leu Thr Glu Pro GlySer Lys Ser Thr Pro Pro 360 365 370 TTC TCT GAA CCC CTG GAG GTG GGG GAGAAT GAC AGT TTA AGC CAG TGC 1205 Phe Ser Glu Pro Leu Glu Val Gly Glu AsnAsp Ser Leu Ser Gln Cys 375 380 385 TTC ACG GGG ACA CAG AGC ACA GTG GGTTCA GAA AGC TGC AAC TGC ACT 1253 Phe Thr Gly Thr Gln Ser Thr Val Gly SerGlu Ser Cys Asn Cys Thr 390 395 400 405 GAG CCC CTG TGC AGG ACT GAT TGGACT CCC ATG TCC TCT GAA AAC TAC 1301 Glu Pro Leu Cys Arg Thr Asp Trp ThrPro Met Ser Ser Glu Asn Tyr 410 415 420 TTG CAA AAA GAG GTG GAC AGT GGCCAT TGC CCG CAC TGG GCA GCC AGC 1349 Leu Gln Lys Glu Val Asp Ser Gly HisCys Pro His Trp Ala Ala Ser 425 430 435 CCC AGC CCC AAC TGG GCA GAT GTCTGC ACA GGC TGC CGG AAC 1391 Pro Ser Pro Asn Trp Ala Asp Val Cys Thr GlyCys Arg Asn 440 445 450 451 amino acids amino acid linear protein notprovided 4 Met Ala Pro Arg Ala Arg Arg Arg Arg Pro Leu Phe Ala Leu LeuLeu 1 5 10 15 Leu Cys Ala Leu Leu Ala Arg Leu Gln Val Ala Leu Gln IleAla Pro 20 25 30 Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg CysCys Asn 35 40 45 Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr ThrThr Ser 50 55 60 Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu AspSer Trp 65 70 75 80 Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys AspThr Gly Lys 85 90 95 Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr ProArg Arg Cys 100 105 110 Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp CysGlu Cys Cys Arg 115 120 125 Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly AlaGln His Pro Leu Gln 130 135 140 Leu Asn Lys Asp Thr Val Cys Lys Pro CysLeu Ala Gly Tyr Phe Ser 145 150 155 160 Asp Ala Phe Ser Ser Thr Asp LysCys Arg Pro Trp Thr Asn Cys Thr 165 170 175 Phe Leu Gly Lys Arg Val GluHis His Gly Thr Glu Lys Ser Asp Ala 180 185 190 Val Cys Ser Ser Ser LeuPro Ala Arg Lys Pro Pro Asn Glu Pro His 195 200 205 Val Tyr Leu Pro GlyLeu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala 210 215 220 Leu Val Ala AlaIle Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys 225 230 235 240 Ala LeuThr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg 245 250 255 LeuSer Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His 260 265 270Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu 275 280285 Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln 290295 300 Gly Gly Val Cys Gln Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln305 310 315 320 Gly Glu Asp Ala Arg Met Leu Ser Leu Val Ser Lys Thr GluIle Glu 325 330 335 Glu Asp Ser Phe Arg Gln Met Pro Thr Glu Asp Glu TyrMet Asp Arg 340 345 350 Pro Ser Gln Pro Thr Asp Gln Leu Leu Phe Leu ThrGlu Pro Gly Ser 355 360 365 Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu GluVal Gly Glu Asn Asp 370 375 380 Ser Leu Ser Gln Cys Phe Thr Gly Thr GlnSer Thr Val Gly Ser Glu 385 390 395 400 Ser Cys Asn Cys Thr Glu Pro LeuCys Arg Thr Asp Trp Thr Pro Met 405 410 415 Ser Ser Glu Asn Tyr Leu GlnLys Glu Val Asp Ser Gly His Cys Pro 420 425 430 His Trp Ala Ala Ser ProSer Pro Asn Trp Ala Asp Val Cys Thr Gly 435 440 445 Cys Arg Asn 450 3136base pairs nucleic acid single linear cDNA NO NO HOMO SAPIENSBONE-MARROW DERIVED DENDRITIC CELLS FULL LENGTH RANK CDS 39..1886 5CCGCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCGCC ATG GCC CCG CGC GCC 53 Met AlaPro Arg Ala 1 5 CGG CGG CGC CGC CCG CTG TTC GCG CTG CTG CTG CTC TGC GCGCTG CTC 101 Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu Leu Cys Ala LeuLeu 10 15 20 GCC CGG CTG CAG GTG GCT TTG CAG ATC GCT CCT CCA TGT ACC AGTGAG 149 Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro Pro Cys Thr Ser Glu25 30 35 AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGT GAA CCA GGA197 Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly 4045 50 AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTA TGT CTG245 Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu 5560 65 CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GAT AAA293 Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys 7075 80 85 TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCC GTG341 Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val 9095 100 GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCT GGG389 Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly 105110 115 TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAG TGC437 Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys 120125 130 GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GAC ACA485 Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr 135140 145 GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCC TCC533 Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe Ser Ser 150155 160 165 ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGA AAGAGA 581 Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg170 175 180 GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCG GTT TGC AGT TCTTCT 629 Val Glu His His Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser185 190 195 CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CAT GTT TAC TTG CCCGGT 677 Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly200 205 210 TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCC CTG GTG GCT GCCATC 725 Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile215 220 225 ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAA GCA CTC ACA GCTAAT 773 Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr Ala Asn230 235 240 245 TTG TGG CAC TGG ATC AAT GAG GCT TGT GGC CGC CTA AGT GGAGAT AAG 821 Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu Ser Gly AspLys 250 255 260 GAG TCC TCA GGT GAC AGT TGT GTC AGT ACA CAC ACG GCA AACTTT GGT 869 Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His Thr Ala Asn PheGly 265 270 275 CAG CAG GGA GCA TGT GAA GGT GTC TTA CTG CTG ACT CTG GAGGAG AAG 917 Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu Thr Leu Glu GluLys 280 285 290 ACA TTT CCA GAA GAT ATG TGC TAC CCA GAT CAA GGT GGT GTCTGT CAG 965 Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln Gly Gly Val CysGln 295 300 305 GGC ACG TGT GTA GGA GGT GGT CCC TAC GCA CAA GGC GAA GATGCC AGG 1013 Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp AlaArg 310 315 320 325 ATG CTC TCA TTG GTC AGC AAG ACC GAG ATA GAG GAA GACAGC TTC AGA 1061 Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu Asp SerPhe Arg 330 335 340 CAG ATG CCC ACA GAA GAT GAA TAC ATG GAC AGG CCC TCCCAG CCC ACA 1109 Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser GlnPro Thr 345 350 355 GAC CAG TTA CTG TTC CTC ACT GAG CCT GGA AGC AAA TCCACA CCT CCT 1157 Asp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser Lys Ser ThrPro Pro 360 365 370 TTC TCT GAA CCC CTG GAG GTG GGG GAG AAT GAC AGT TTAAGC CAG TGC 1205 Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu SerGln Cys 375 380 385 TTC ACG GGG ACA CAG AGC ACA GTG GGT TCA GAA AGC TGCAAC TGC ACT 1253 Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu Ser Cys AsnCys Thr 390 395 400 405 GAG CCC CTG TGC AGG ACT GAT TGG ACT CCC ATG TCCTCT GAA AAC TAC 1301 Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser SerGlu Asn Tyr 410 415 420 TTG CAA AAA GAG GTG GAC AGT GGC CAT TGC CCG CACTGG GCA GCC AGC 1349 Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro His TrpAla Ala Ser 425 430 435 CCC AGC CCC AAC TGG GCA GAT GTC TGC ACA GGC TGCCGG AAC CCT CCT 1397 Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly Cys ArgAsn Pro Pro 440 445 450 GGG GAG GAC TGT GAA CCC CTC GTG GGT TCC CCA AAACGT GGA CCC TTG 1445 Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro Lys ArgGly Pro Leu 455 460 465 CCC CAG TGC GCC TAT GGC ATG GGC CTT CCC CCT GAAGAA GAA GCC AGC 1493 Pro Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro Glu GluGlu Ala Ser 470 475 480 485 AGG ACG GAG GCC AGA GAC CAG CCC GAG GAT GGGGCT GAT GGG AGG CTC 1541 Arg Thr Glu Ala Arg Asp Gln Pro Glu Asp Gly AlaAsp Gly Arg Leu 490 495 500 CCA AGC TCA GCG AGG GCA GGT GCC GGG TCT GGAAGC TCC CCT GGT GGC 1589 Pro Ser Ser Ala Arg Ala Gly Ala Gly Ser Gly SerSer Pro Gly Gly 505 510 515 CAG TCC CCT GCA TCT GGA AAT GTG ACT GGA AACAGT AAC TCC ACG TTC 1637 Gln Ser Pro Ala Ser Gly Asn Val Thr Gly Asn SerAsn Ser Thr Phe 520 525 530 ATC TCC AGC GGG CAG GTG ATG AAC TTC AAG GGCGAC ATC ATC GTG GTC 1685 Ile Ser Ser Gly Gln Val Met Asn Phe Lys Gly AspIle Ile Val Val 535 540 545 TAC GTC AGC CAG ACC TCG CAG GAG GGC GCG GCGGCG GCT GCG GAG CCC 1733 Tyr Val Ser Gln Thr Ser Gln Glu Gly Ala Ala AlaAla Ala Glu Pro 550 555 560 565 ATG GGC CGC CCG GTG CAG GAG GAG ACC CTGGCG CGC CGA GAC TCC TTC 1781 Met Gly Arg Pro Val Gln Glu Glu Thr Leu AlaArg Arg Asp Ser Phe 570 575 580 GCG GGG AAC GGC CCG CGC TTC CCG GAC CCGTGC GGC GGC CCC GAG GGG 1829 Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro CysGly Gly Pro Glu Gly 585 590 595 CTG CGG GAG CCG GAG AAG GCC TCG AGG CCGGTG CAG GAG CAA GGC GGG 1877 Leu Arg Glu Pro Glu Lys Ala Ser Arg Pro ValGln Glu Gln Gly Gly 600 605 610 GCC AAG GCT TGAGCGCCCC CCATGGCTGGGAGCCCGAAG CTCGGAGCCA 1926 Ala Lys Ala 615 GGGCTCGCGA GGGCAGCACCGCAGCCTCTG CCCCAGCCCC GGCCACCCAG GGATCGATCG 1986 GTACAGTCGA GGAAGACCACCCGGCATTCT CTGCCCACTT TGCCTTCCAG GAAATGGGCT 2046 TTTCAGGAAG TGAATTGATGAGGACTGTCC CCATGCCCAC GGATGCTCAG CAGCCCGCCG 2106 CACTGGGGCA GATGTCTCCCCTGCCACTCC TCAAACTCGC AGCAGTAATT TGTGGCACTA 2166 TGACAGCTAT TTTTATGACTATCCTGTTCT GTGGGGGGGG GGTCTATGTT TTCCCCCCAT 2226 ATTTGTATTC CTTTTCATAACTTTTCTTGA TATCTTTCCT CCCTCTTTTT TAATGTAAAG 2286 GTTTTCTCAA AAATTCTCCTAAAGGTGAGG GTCTCTTTCT TTTCTCTTTT CCTTTTTTTT 2346 TTCTTTTTTT GGCAACCTGGCTCTGGCCCA GGCTAGAGTG CAGTGGTGCG ATTATAGCCC 2406 GGTGCAGCCT CTAACTCCTGGGCTCAAGCA ATCCAAGTGA TCCTCCCACC TCAACCTTCG 2466 GAGTAGCTGG GATCACAGCTGCAGGCCACG CCCAGCTTCC TCCCCCCGAC TCCCCCCCCC 2526 CAGAGACACG GTCCCACCATGTTACCCAGC CTGGTCTCAA ACTCCCCAGC TAAAGCAGTC 2586 CTCCAGCCTC GGCCTCCCAAAGTACTGGGA TTACAGGCGT GAGCCCCCAC GCTGGCCTGC 2646 TTTACGTATT TTCTTTTGTGCCCCTGCTCA CAGTGTTTTA GAGATGGCTT TCCCAGTGTG 2706 TGTTCATTGT AAACACTTTTGGGAAAGGGC TAAACATGTG AGGCCTGGAG ATAGTTGCTA 2766 AGTTGCTAGG AACATGTGGTGGGACTTTCA TATTCTGAAA AATGTTCTAT ATTCTCATTT 2826 TTCTAAAAGA AAGAAAAAAGGAAACCCGAT TTATTTCTCC TGAATCTTTT TAAGTTTGTG 2886 TCGTTCCTTA AGCAGAACTAAGCTCAGTAT GTGACCTTAC CCGCTAGGTG GTTAATTTAT 2946 CCATGCTGGC AGAGGCACTCAGGTACTTGG TAAGCAAATT TCTAAAACTC CAAGTTGCTG 3006 CAGCTTGGCA TTCTTCTTATTCTAGAGGTC TCTCTGGAAA AGATGGAGAA AATGAACAGG 3066 ACATGGGGCT CCTGGAAAGAAAGGGCCCGG GAAGTTCAAG GAAGAATAAA GTTGAAATTT 3126 TAAAAAAAAA 3136 616amino acids amino acid linear protein not provided 6 Met Ala Pro Arg AlaArg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu 1 5 10 15 Leu Cys Ala LeuLeu Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro 20 25 30 Pro Cys Thr SerGlu Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn 35 40 45 Lys Cys Glu ProGly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser 50 55 60 Asp Ser Val CysLeu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 65 70 75 80 Asn Glu GluAsp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys 85 90 95 Ala Leu ValAla Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys 100 105 110 Ala CysThr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 115 120 125 ArgAsn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln 130 135 140Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 145 150155 160 Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr165 170 175 Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser AspAla 180 185 190 Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn GluPro His 195 200 205 Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe AlaSer Val Ala 210 215 220 Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr ArgLys Lys Gly Lys 225 230 235 240 Ala Leu Thr Ala Asn Leu Trp His Trp IleAsn Glu Ala Cys Gly Arg 245 250 255 Leu Ser Gly Asp Lys Glu Ser Ser GlyAsp Ser Cys Val Ser Thr His 260 265 270 Thr Ala Asn Phe Gly Gln Gln GlyAla Cys Glu Gly Val Leu Leu Leu 275 280 285 Thr Leu Glu Glu Lys Thr PhePro Glu Asp Met Cys Tyr Pro Asp Gln 290 295 300 Gly Gly Val Cys Gln GlyThr Cys Val Gly Gly Gly Pro Tyr Ala Gln 305 310 315 320 Gly Glu Asp AlaArg Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu 325 330 335 Glu Asp SerPhe Arg Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg 340 345 350 Pro SerGln Pro Thr Asp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser 355 360 365 LysSer Thr Pro Pro Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp 370 375 380Ser Leu Ser Gln Cys Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu 385 390395 400 Ser Cys Asn Cys Thr Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met405 410 415 Ser Ser Glu Asn Tyr Leu Gln Lys Glu Val Asp Ser Gly His CysPro 420 425 430 His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp Val CysThr Gly 435 440 445 Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu ValGly Ser Pro 450 455 460 Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly MetGly Leu Pro Pro 465 470 475 480 Glu Glu Glu Ala Ser Arg Thr Glu Ala ArgAsp Gln Pro Glu Asp Gly 485 490 495 Ala Asp Gly Arg Leu Pro Ser Ser AlaArg Ala Gly Ala Gly Ser Gly 500 505 510 Ser Ser Pro Gly Gly Gln Ser ProAla Ser Gly Asn Val Thr Gly Asn 515 520 525 Ser Asn Ser Thr Phe Ile SerSer Gly Gln Val Met Asn Phe Lys Gly 530 535 540 Asp Ile Ile Val Val TyrVal Ser Gln Thr Ser Gln Glu Gly Ala Ala 545 550 555 560 Ala Ala Ala GluPro Met Gly Arg Pro Val Gln Glu Glu Thr Leu Ala 565 570 575 Arg Arg AspSer Phe Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro Cys 580 585 590 Gly GlyPro Glu Gly Leu Arg Glu Pro Glu Lys Ala Ser Arg Pro Val 595 600 605 GlnGlu Gln Gly Gly Ala Lys Ala 610 615 8 amino acids amino acid notrelevant linear peptide not provided FLAG_ peptide 7 Asp Tyr Lys Asp AspAsp Asp Lys 1 5 232 amino acids amino acid not relevant linear proteinHuman IgG1 Fc mutein 8 Glu Pro Arg Ser Cys Asp Lys Thr His Thr Cys ProPro Cys Pro Ala 1 5 10 15 Pro Glu Ala Glu Gly Ala Pro Ser Val Phe LeuPhe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro GluVal Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val LysPhe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr LysPro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Asp Tyr Lys CysLys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Met Gln Lys Thr IleSer Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr LeuPro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu ThrCys Leu Val Lys Gly Phe Tyr Pro Arg 145 150 155 160 His Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys LeuThr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser CysSer Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 SerLeu Ser Leu Ser Pro Gly Lys 225 230 31 amino acids amino acid notrelevant linear peptide NO NO CMV (R2780 Leader) Met1-Arg28 is theactual leader peptide; Arg29 strengthens the furin cleavage site;nucleotides encoding eThr30 and Ser31 add a Spe1 site. 9 Met Ala Arg ArgLeu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr 1 5 10 15 Val Ala LeuAla Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser 20 25 30 1630 base pairsnucleic acid single linear cDNA NO NO Mus musculus <Unknown> RANKL CDS3..884 10 CC GGC GTC CCA CAC GAG GGT CCG CTG CAC CCC GCG CCT TCT GCA CCG47 Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser Ala Pro 1 5 10 15GCT CCG GCG CCG CCA CCC GCC GCC TCC CGC TCC ATG TTC CTG GCC CTC 95 AlaPro Ala Pro Pro Pro Ala Ala Ser Arg Ser Met Phe Leu Ala Leu 20 25 30 CTGGGG CTG GGA CTG GGC CAG GTG GTC TGC AGC ATC GCT CTG TTC CTG 143 Leu GlyLeu Gly Leu Gly Gln Val Val Cys Ser Ile Ala Leu Phe Leu 35 40 45 TAC TTTCGA GCG CAG ATG GAT CCT AAC AGA ATA TCA GAA GAC AGC ACT 191 Tyr Phe ArgAla Gln Met Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr 50 55 60 CAC TGC TTTTAT AGA ATC CTG AGA CTC CAT GAA AAC GCA GAT TTG CAG 239 His Cys Phe TyrArg Ile Leu Arg Leu His Glu Asn Ala Asp Leu Gln 65 70 75 GAC TCG ACT CTGGAG AGT GAA GAC ACA CTA CCT GAC TCC TGC AGG AGG 287 Asp Ser Thr Leu GluSer Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg 80 85 90 95 ATG AAA CAA GCCTTT CAG GGG GCC GTG CAG AAG GAA CTG CAA CAC ATT 335 Met Lys Gln Ala PheGln Gly Ala Val Gln Lys Glu Leu Gln His Ile 100 105 110 GTG GGG CCA CAGCGC TTC TCA GGA GCT CCA GCT ATG ATG GAA GGC TCA 383 Val Gly Pro Gln ArgPhe Ser Gly Ala Pro Ala Met Met Glu Gly Ser 115 120 125 TGG TTG GAT GTGGCC CAG CGA GGC AAG CCT GAG GCC CAG CCA TTT GCA 431 Trp Leu Asp Val AlaGln Arg Gly Lys Pro Glu Ala Gln Pro Phe Ala 130 135 140 CAC CTC ACC ATCAAT GCT GCC AGC ATC CCA TCG GGT TCC CAT AAA GTC 479 His Leu Thr Ile AsnAla Ala Ser Ile Pro Ser Gly Ser His Lys Val 145 150 155 ACT CTG TCC TCTTGG TAC CAC GAT CGA GGC TGG GCC AAG ATC TCT AAC 527 Thr Leu Ser Ser TrpTyr His Asp Arg Gly Trp Ala Lys Ile Ser Asn 160 165 170 175 ATG ACG TTAAGC AAC GGA AAA CTA AGG GTT AAC CAA GAT GGC TTC TAT 575 Met Thr Leu SerAsn Gly Lys Leu Arg Val Asn Gln Asp Gly Phe Tyr 180 185 190 TAC CTG TACGCC AAC ATT TGC TTT CGG CAT CAT GAA ACA TCG GGA AGC 623 Tyr Leu Tyr AlaAsn Ile Cys Phe Arg His His Glu Thr Ser Gly Ser 195 200 205 GTA CCT ACAGAC TAT CTT CAG CTG ATG GTG TAT GTC GTT AAA ACC AGC 671 Val Pro Thr AspTyr Leu Gln Leu Met Val Tyr Val Val Lys Thr Ser 210 215 220 ATC AAA ATCCCA AGT TCT CAT AAC CTG ATG AAA GGA GGG AGC ACG AAA 719 Ile Lys Ile ProSer Ser His Asn Leu Met Lys Gly Gly Ser Thr Lys 225 230 235 AAC TGG TCGGGC AAT TCT GAA TTC CAC TTT TAT TCC ATA AAT GTT GGG 767 Asn Trp Ser GlyAsn Ser Glu Phe His Phe Tyr Ser Ile Asn Val Gly 240 245 250 255 GGA TTTTTC AAG CTC CGA GCT GGT GAA GAA ATT AGC ATT CAG GTG TCC 815 Gly Phe PheLys Leu Arg Ala Gly Glu Glu Ile Ser Ile Gln Val Ser 260 265 270 AAC CCTTCC CTG CTG GAT CCG GAT CAA GAT GCG ACG TAC TTT GGG GCT 863 Asn Pro SerLeu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala 275 280 285 TTC AAAGTT CAG GAC ATA GAC TGAGACTCAT TTCGTGGAAC ATTAGCATGG 914 Phe Lys Val GlnAsp Ile Asp 290 ATGTCCTAGA TGTTTGGAAA CTTCTTAAAA AATGGATGAT GTCTATACATGTGTAAGACT 974 ACTAAGAGAC ATGGCCCACG GTGTATGAAA CTCACAGCCC TCTCTCTTGAGCCTGTACAG 1034 GTTGTGTATA TGTAAAGTCC ATAGGTGATG TTAGATTCAT GGTGATTACACAACGGTTTT 1094 ACAATTTTGT AATGATTTCC TAGAATTGAA CCAGATTGGG AGAGGTATTCCGATGCTTAT 1154 GAAAAACTTA CACGTGAGCT ATGGAAGGGG GTCACAGTCT CTGGGTCTAACCCCTGGACA 1214 TGTGCCACTG AGAACCTTGA AATTAAGAGG ATGCCATGTC ATTGCAAAGAAATGATAGTG 1274 TGAAGGGTTA AGTTCTTTTG AATTGTTACA TTGCGCTGGG ACCTGCAAATAAGTTCTTTT 1334 TTTCTAATGA GGAGAGAAAA ATATATGTAT TTTTATATAA TGTCTAAAGTTATATTTCAG 1394 GTGTAATGTT TTCTGTGCAA AGTTTTGTAA ATTATATTTG TGCTATAGTATTTGATTCAA 1454 AATATTTAAA AATGTCTCAC TGTTGACATA TTTAATGTTT TAAATGTACAGATGTATTTA 1514 ACTGGTGCAC TTTGTAATTC CCCTGAAGGT ACTCGTAGCT AAGGGGGCAGAATACTGTTT 1574 CTGGTGACCA CATGTAGTTT ATTTCTTTAT TCTTTTTAAC TTAATAGAGTCTTCAG 1630 294 amino acids amino acid linear protein not provided 11Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser Ala Pro Ala 1 5 1015 Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser Met Phe Leu Ala Leu Leu 20 2530 Gly Leu Gly Leu Gly Gln Val Val Cys Ser Ile Ala Leu Phe Leu Tyr 35 4045 Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr His 50 5560 Cys Phe Tyr Arg Ile Leu Arg Leu His Glu Asn Ala Asp Leu Gln Asp 65 7075 80 Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg Met 8590 95 Lys Gln Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val100 105 110 Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu Gly SerTrp 115 120 125 Leu Asp Val Ala Gln Arg Gly Lys Pro Glu Ala Gln Pro PheAla His 130 135 140 Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser Gly Ser HisLys Val Thr 145 150 155 160 Leu Ser Ser Trp Tyr His Asp Arg Gly Trp AlaLys Ile Ser Asn Met 165 170 175 Thr Leu Ser Asn Gly Lys Leu Arg Val AsnGln Asp Gly Phe Tyr Tyr 180 185 190 Leu Tyr Ala Asn Ile Cys Phe Arg HisHis Glu Thr Ser Gly Ser Val 195 200 205 Pro Thr Asp Tyr Leu Gln Leu MetVal Tyr Val Val Lys Thr Ser Ile 210 215 220 Lys Ile Pro Ser Ser His AsnLeu Met Lys Gly Gly Ser Thr Lys Asn 225 230 235 240 Trp Ser Gly Asn SerGlu Phe His Phe Tyr Ser Ile Asn Val Gly Gly 245 250 255 Phe Phe Lys LeuArg Ala Gly Glu Glu Ile Ser Ile Gln Val Ser Asn 260 265 270 Pro Ser LeuLeu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala Phe 275 280 285 Lys ValGln Asp Ile Asp 290 954 base pairs nucleic acid single linear cDNA NO NOHomo sapiens <Unknown> huRANKL (full length) CDS 1..951 12 ATG CGC CGCGCC AGC AGA GAC TAC ACC AAG TAC CTG CGT GGC TCG GAG 48 Met Arg Arg AlaSer Arg Asp Tyr Thr Lys Tyr Leu Arg Gly Ser Glu 1 5 10 15 GAG ATG GGCGGC GGC CCC GGA GCC CCG CAC GAG GGC CCC CTG CAC GCC 96 Glu Met Gly GlyGly Pro Gly Ala Pro His Glu Gly Pro Leu His Ala 20 25 30 CCG CCG CCG CCTGCG CCG CAC CAG CCC CCC GCC GCC TCC CGC TCC ATG 144 Pro Pro Pro Pro AlaPro His Gln Pro Pro Ala Ala Ser Arg Ser Met 35 40 45 TTC GTG GCC CTC CTGGGG CTG GGG CTG GGC CAG GTT GTC TGC AGC GTC 192 Phe Val Ala Leu Leu GlyLeu Gly Leu Gly Gln Val Val Cys Ser Val 50 55 60 GCC CTG TTC TTC TAT TTCAGA GCG CAG ATG GAT CCT AAT AGA ATA TCA 240 Ala Leu Phe Phe Tyr Phe ArgAla Gln Met Asp Pro Asn Arg Ile Ser 65 70 75 80 GAA GAT GGC ACT CAC TGCATT TAT AGA ATT TTG AGA CTC CAT GAA AAT 288 Glu Asp Gly Thr His Cys IleTyr Arg Ile Leu Arg Leu His Glu Asn 85 90 95 GCA GAT TTT CAA GAC ACA ACTCTG GAG AGT CAA GAT ACA AAA TTA ATA 336 Ala Asp Phe Gln Asp Thr Thr LeuGlu Ser Gln Asp Thr Lys Leu Ile 100 105 110 CCT GAT TCA TGT AGG AGA ATTAAA CAG GCC TTT CAA GGA GCT GTG CAA 384 Pro Asp Ser Cys Arg Arg Ile LysGln Ala Phe Gln Gly Ala Val Gln 115 120 125 AAG GAA TTA CAA CAT ATC GTTGGA TCA CAG CAC ATC AGA GCA GAG AAA 432 Lys Glu Leu Gln His Ile Val GlySer Gln His Ile Arg Ala Glu Lys 130 135 140 GCG ATG GTG GAT GGC TCA TGGTTA GAT CTG GCC AAG AGG AGC AAG CTT 480 Ala Met Val Asp Gly Ser Trp LeuAsp Leu Ala Lys Arg Ser Lys Leu 145 150 155 160 GAA GCT CAG CCT TTT GCTCAT CTC ACT ATT AAT GCC ACC GAC ATC CCA 528 Glu Ala Gln Pro Phe Ala HisLeu Thr Ile Asn Ala Thr Asp Ile Pro 165 170 175 TCT GGT TCC CAT AAA GTGAGT CTG TCC TCT TGG TAC CAT GAT CGG GGT 576 Ser Gly Ser His Lys Val SerLeu Ser Ser Trp Tyr His Asp Arg Gly 180 185 190 TGG GCC AAG ATC TCC AACATG ACT TTT AGC AAT GGA AAA CTA ATA GTT 624 Trp Ala Lys Ile Ser Asn MetThr Phe Ser Asn Gly Lys Leu Ile Val 195 200 205 AAT CAG GAT GGC TTT TATTAC CTG TAT GCC AAC ATT TGC TTT CGA CAT 672 Asn Gln Asp Gly Phe Tyr TyrLeu Tyr Ala Asn Ile Cys Phe Arg His 210 215 220 CAT GAA ACT TCA GGA GACCTA GCT ACA GAG TAT CTT CAA CTA ATG GTG 720 His Glu Thr Ser Gly Asp LeuAla Thr Glu Tyr Leu Gln Leu Met Val 225 230 235 240 TAC GTC ACT AAA ACCAGC ATC AAA ATC CCA AGT TCT CAT ACC CTG ATG 768 Tyr Val Thr Lys Thr SerIle Lys Ile Pro Ser Ser His Thr Leu Met 245 250 255 AAA GGA GGA AGC ACCAAG TAT TGG TCA GGG AAT TCT GAA TTC CAT TTT 816 Lys Gly Gly Ser Thr LysTyr Trp Ser Gly Asn Ser Glu Phe His Phe 260 265 270 TAT TCC ATA AAC GTTGGT GGA TTT TTT AAG TTA CGG TCT GGA GAG GAA 864 Tyr Ser Ile Asn Val GlyGly Phe Phe Lys Leu Arg Ser Gly Glu Glu 275 280 285 ATC AGC ATC GAG GTCTCC AAC CCC TCC TTA CTG GAT CCG GAT CAG GAT 912 Ile Ser Ile Glu Val SerAsn Pro Ser Leu Leu Asp Pro Asp Gln Asp 290 295 300 GCA ACA TAC TTT GGGGCT TTT AAA GTT CGA GAT ATA GAT TGA 954 Ala Thr Tyr Phe Gly Ala Phe LysVal Arg Asp Ile Asp 305 310 315 317 amino acids amino acid linearprotein not provided 13 Met Arg Arg Ala Ser Arg Asp Tyr Thr Lys Tyr LeuArg Gly Ser Glu 1 5 10 15 Glu Met Gly Gly Gly Pro Gly Ala Pro His GluGly Pro Leu His Ala 20 25 30 Pro Pro Pro Pro Ala Pro His Gln Pro Pro AlaAla Ser Arg Ser Met 35 40 45 Phe Val Ala Leu Leu Gly Leu Gly Leu Gly GlnVal Val Cys Ser Val 50 55 60 Ala Leu Phe Phe Tyr Phe Arg Ala Gln Met AspPro Asn Arg Ile Ser 65 70 75 80 Glu Asp Gly Thr His Cys Ile Tyr Arg IleLeu Arg Leu His Glu Asn 85 90 95 Ala Asp Phe Gln Asp Thr Thr Leu Glu SerGln Asp Thr Lys Leu Ile 100 105 110 Pro Asp Ser Cys Arg Arg Ile Lys GlnAla Phe Gln Gly Ala Val Gln 115 120 125 Lys Glu Leu Gln His Ile Val GlySer Gln His Ile Arg Ala Glu Lys 130 135 140 Ala Met Val Asp Gly Ser TrpLeu Asp Leu Ala Lys Arg Ser Lys Leu 145 150 155 160 Glu Ala Gln Pro PheAla His Leu Thr Ile Asn Ala Thr Asp Ile Pro 165 170 175 Ser Gly Ser HisLys Val Ser Leu Ser Ser Trp Tyr His Asp Arg Gly 180 185 190 Trp Ala LysIle Ser Asn Met Thr Phe Ser Asn Gly Lys Leu Ile Val 195 200 205 Asn GlnAsp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 210 215 220 HisGlu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met Val 225 230 235240 Tyr Val Thr Lys Thr Ser Ile Lys Ile Pro Ser Ser His Thr Leu Met 245250 255 Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu Phe His Phe260 265 270 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ser Gly GluGlu 275 280 285 Ile Ser Ile Glu Val Ser Asn Pro Ser Leu Leu Asp Pro AspGln Asp 290 295 300 Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp Ile Asp305 310 315 1878 base pairs nucleic acid single linear cDNA NO NO MurineMurine Fetal Liver Epithelium muRANK CDS 1..1875 14 ATG GCC CCG CGC GCCCGG CGG CGC CGC CAG CTG CCC GCG CCG CTG CTG 48 Met Ala Pro Arg Ala ArgArg Arg Arg Gln Leu Pro Ala Pro Leu Leu 1 5 10 15 GCG CTC TGC GTG CTGCTC GTT CCA CTG CAG GTG ACT CTC CAG GTC ACT 96 Ala Leu Cys Val Leu LeuVal Pro Leu Gln Val Thr Leu Gln Val Thr 20 25 30 CCT CCA TGC ACC CAG GAGAGG CAT TAT GAG CAT CTC GGA CGG TGT TGC 144 Pro Pro Cys Thr Gln Glu ArgHis Tyr Glu His Leu Gly Arg Cys Cys 35 40 45 AGC AGA TGC GAA CCA GGA AAGTAC CTG TCC TCT AAG TGC ACT CCT ACC 192 Ser Arg Cys Glu Pro Gly Lys TyrLeu Ser Ser Lys Cys Thr Pro Thr 50 55 60 TCC GAC AGT GTG TGT CTG CCC TGTGGC CCC GAT GAG TAC TTG GAC ACC 240 Ser Asp Ser Val Cys Leu Pro Cys GlyPro Asp Glu Tyr Leu Asp Thr 65 70 75 80 TGG AAT GAA GAA GAT AAA TGC TTGCTG CAT AAA GTC TGT GAT GCA GGC 288 Trp Asn Glu Glu Asp Lys Cys Leu LeuHis Lys Val Cys Asp Ala Gly 85 90 95 AAG GCC CTG GTG GCG GTG GAT CCT GGCAAC CAC ACG GCC CCG CGT CGC 336 Lys Ala Leu Val Ala Val Asp Pro Gly AsnHis Thr Ala Pro Arg Arg 100 105 110 TGT GCT TGC ACG GCT GGC TAC CAC TGGAAC TCA GAC TGC GAG TGC TGC 384 Cys Ala Cys Thr Ala Gly Tyr His Trp AsnSer Asp Cys Glu Cys Cys 115 120 125 CGC AGG AAC ACG GAG TGT GCA CCT GGCTTC GGA GCT CAG CAT CCC TTG 432 Arg Arg Asn Thr Glu Cys Ala Pro Gly PheGly Ala Gln His Pro Leu 130 135 140 CAG CTC AAC AAG GAT ACG GTG TGC ACACCC TGC CTC CTG GGC TTC TTC 480 Gln Leu Asn Lys Asp Thr Val Cys Thr ProCys Leu Leu Gly Phe Phe 145 150 155 160 TCA GAT GTC TTT TCG TCC ACA GACAAA TGC AAA CCT TGG ACC AAC TGC 528 Ser Asp Val Phe Ser Ser Thr Asp LysCys Lys Pro Trp Thr Asn Cys 165 170 175 ACC CTC CTT GGA AAG CTA GAA GCACAC CAG GGG ACA ACG GAA TCA GAT 576 Thr Leu Leu Gly Lys Leu Glu Ala HisGln Gly Thr Thr Glu Ser Asp 180 185 190 GTG GTC TGC AGC TCT TCC ATG ACACTG AGG AGA CCA CCC AAG GAG GCC 624 Val Val Cys Ser Ser Ser Met Thr LeuArg Arg Pro Pro Lys Glu Ala 195 200 205 CAG GCT TAC CTG CCC AGT CTC ATCGTT CTG CTC CTC TTC ATC TCT GTG 672 Gln Ala Tyr Leu Pro Ser Leu Ile ValLeu Leu Leu Phe Ile Ser Val 210 215 220 GTA GTA GTG GCT GCC ATC ATC TTCGGC GTT TAC TAC AGG AAG GGA GGG 720 Val Val Val Ala Ala Ile Ile Phe GlyVal Tyr Tyr Arg Lys Gly Gly 225 230 235 240 AAA GCG CTG ACA GCT AAT TTGTGG AAT TGG GTC AAT GAT GCT TGC AGT 768 Lys Ala Leu Thr Ala Asn Leu TrpAsn Trp Val Asn Asp Ala Cys Ser 245 250 255 AGT CTA AGT GGA AAT AAG GAGTCC TCA GGG GAC CGT TGT GCT GGT TCC 816 Ser Leu Ser Gly Asn Lys Glu SerSer Gly Asp Arg Cys Ala Gly Ser 260 265 270 CAC TCG GCA ACC TCC AGT CAGCAA GAA GTG TGT GAA GGT ATC TTA CTA 864 His Ser Ala Thr Ser Ser Gln GlnGlu Val Cys Glu Gly Ile Leu Leu 275 280 285 ATG ACT CGG GAG GAG AAG ATGGTT CCA GAA GAC GGT GCT GGA GTC TGT 912 Met Thr Arg Glu Glu Lys Met ValPro Glu Asp Gly Ala Gly Val Cys 290 295 300 GGG CCT GTG TGT GCG GCA GGTGGG CCC TGG GCA GAA GTC AGA GAT TCT 960 Gly Pro Val Cys Ala Ala Gly GlyPro Trp Ala Glu Val Arg Asp Ser 305 310 315 320 AGG ACG TTC ACA CTG GTCAGC GAG GTT GAG ACG CAA GGA GAC CTC TCG 1008 Arg Thr Phe Thr Leu Val SerGlu Val Glu Thr Gln Gly Asp Leu Ser 325 330 335 AGG AAG ATT CCC ACA GAGGAT GAG TAC ACG GAC CGG CCC TCG CAG CCT 1056 Arg Lys Ile Pro Thr Glu AspGlu Tyr Thr Asp Arg Pro Ser Gln Pro 340 345 350 TCG ACT GGT TCA CTG CTCCTA ATC CAG CAG GGA AGC AAA TCT ATA CCC 1104 Ser Thr Gly Ser Leu Leu LeuIle Gln Gln Gly Ser Lys Ser Ile Pro 355 360 365 CCA TTC CAG GAG CCC CTGGAA GTG GGG GAG AAC GAC AGT TTA AGC CAG 1152 Pro Phe Gln Glu Pro Leu GluVal Gly Glu Asn Asp Ser Leu Ser Gln 370 375 380 TGT TTC ACC GGG ACT GAAAGC ACG GTG GAT TCT GAG GGC TGT GAC TTC 1200 Cys Phe Thr Gly Thr Glu SerThr Val Asp Ser Glu Gly Cys Asp Phe 385 390 395 400 ACT GAG CCT CCG AGCAGA ACT GAC TCT ATG CCC GTG TCC CCT GAA AAG 1248 Thr Glu Pro Pro Ser ArgThr Asp Ser Met Pro Val Ser Pro Glu Lys 405 410 415 CAC CTG ACA AAA GAAATA GAA GGT GAC AGT TGC CTC CCC TGG GTG GTC 1296 His Leu Thr Lys Glu IleGlu Gly Asp Ser Cys Leu Pro Trp Val Val 420 425 430 AGC TCC AAC TCA ACAGAT GGC TAC ACA GGC AGT GGG AAC ACT CCT GGG 1344 Ser Ser Asn Ser Thr AspGly Tyr Thr Gly Ser Gly Asn Thr Pro Gly 435 440 445 GAG GAC CAT GAA CCCTTT CCA GGG TCC CTG AAA TGT GGA CCA TTG CCC 1392 Glu Asp His Glu Pro PhePro Gly Ser Leu Lys Cys Gly Pro Leu Pro 450 455 460 CAG TGT GCC TAC AGCATG GGC TTT CCC AGT GAA GCA GCA GCC AGC ATG 1440 Gln Cys Ala Tyr Ser MetGly Phe Pro Ser Glu Ala Ala Ala Ser Met 465 470 475 480 GCA GAG GCG GGAGTA CGG CCC CAG GAC AGG GCT GAT GAG AGG GGA GCC 1488 Ala Glu Ala Gly ValArg Pro Gln Asp Arg Ala Asp Glu Arg Gly Ala 485 490 495 TCA GGG TCC GGGAGC TCC CCC AGT GAC CAG CCA CCT GCC TCT GGG AAC 1536 Ser Gly Ser Gly SerSer Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn 500 505 510 GTG ACT GGA AACAGT AAC TCC ACG TTC ATC TCT AGC GGG CAG GTG ATG 1584 Val Thr Gly Asn SerAsn Ser Thr Phe Ile Ser Ser Gly Gln Val Met 515 520 525 AAC TTC AAG GGTGAC ATC ATC GTG GTG TAT GTC AGC CAG ACC TCG CAG 1632 Asn Phe Lys Gly AspIle Ile Val Val Tyr Val Ser Gln Thr Ser Gln 530 535 540 GAG GGC CCG GGTTCC GCA GAG CCC GAG TCG GAG CCC GTG GGC CGC CCT 1680 Glu Gly Pro Gly SerAla Glu Pro Glu Ser Glu Pro Val Gly Arg Pro 545 550 555 560 GTG CAG GAGGAG ACG CTG GCA CAC AGA GAC TCC TTT GCG GGC ACC GCG 1728 Val Gln Glu GluThr Leu Ala His Arg Asp Ser Phe Ala Gly Thr Ala 565 570 575 CCG CGC TTCCCC GAC GTC TGT GCC ACC GGG GCT GGG CTG CAG GAG CAG 1776 Pro Arg Phe ProAsp Val Cys Ala Thr Gly Ala Gly Leu Gln Glu Gln 580 585 590 GGG GCA CCCCGG CAG AAG GAC GGG ACA TCG CGG CCG GTG CAG GAG CAG 1824 Gly Ala Pro ArgGln Lys Asp Gly Thr Ser Arg Pro Val Gln Glu Gln 595 600 605 GGT GGG GCGCAG ACT TCA CTC CAT ACC CAG GGG TCC GGA CAA TGT GCA 1872 Gly Gly Ala GlnThr Ser Leu His Thr Gln Gly Ser Gly Gln Cys Ala 610 615 620 GAA TGA 1878Glu 625 625 amino acids amino acid linear protein not provided 15 MetAla Pro Arg Ala Arg Arg Arg Arg Gln Leu Pro Ala Pro Leu Leu 1 5 10 15Ala Leu Cys Val Leu Leu Val Pro Leu Gln Val Thr Leu Gln Val Thr 20 25 30Pro Pro Cys Thr Gln Glu Arg His Tyr Glu His Leu Gly Arg Cys Cys 35 40 45Ser Arg Cys Glu Pro Gly Lys Tyr Leu Ser Ser Lys Cys Thr Pro Thr 50 55 60Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Thr 65 70 7580 Trp Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Ala Gly 85 9095 Lys Ala Leu Val Ala Val Asp Pro Gly Asn His Thr Ala Pro Arg Arg 100105 110 Cys Ala Cys Thr Ala Gly Tyr His Trp Asn Ser Asp Cys Glu Cys Cys115 120 125 Arg Arg Asn Thr Glu Cys Ala Pro Gly Phe Gly Ala Gln His ProLeu 130 135 140 Gln Leu Asn Lys Asp Thr Val Cys Thr Pro Cys Leu Leu GlyPhe Phe 145 150 155 160 Ser Asp Val Phe Ser Ser Thr Asp Lys Cys Lys ProTrp Thr Asn Cys 165 170 175 Thr Leu Leu Gly Lys Leu Glu Ala His Gln GlyThr Thr Glu Ser Asp 180 185 190 Val Val Cys Ser Ser Ser Met Thr Leu ArgArg Pro Pro Lys Glu Ala 195 200 205 Gln Ala Tyr Leu Pro Ser Leu Ile ValLeu Leu Leu Phe Ile Ser Val 210 215 220 Val Val Val Ala Ala Ile Ile PheGly Val Tyr Tyr Arg Lys Gly Gly 225 230 235 240 Lys Ala Leu Thr Ala AsnLeu Trp Asn Trp Val Asn Asp Ala Cys Ser 245 250 255 Ser Leu Ser Gly AsnLys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser 260 265 270 His Ser Ala ThrSer Ser Gln Gln Glu Val Cys Glu Gly Ile Leu Leu 275 280 285 Met Thr ArgGlu Glu Lys Met Val Pro Glu Asp Gly Ala Gly Val Cys 290 295 300 Gly ProVal Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg Asp Ser 305 310 315 320Arg Thr Phe Thr Leu Val Ser Glu Val Glu Thr Gln Gly Asp Leu Ser 325 330335 Arg Lys Ile Pro Thr Glu Asp Glu Tyr Thr Asp Arg Pro Ser Gln Pro 340345 350 Ser Thr Gly Ser Leu Leu Leu Ile Gln Gln Gly Ser Lys Ser Ile Pro355 360 365 Pro Phe Gln Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu SerGln 370 375 380 Cys Phe Thr Gly Thr Glu Ser Thr Val Asp Ser Glu Gly CysAsp Phe 385 390 395 400 Thr Glu Pro Pro Ser Arg Thr Asp Ser Met Pro ValSer Pro Glu Lys 405 410 415 His Leu Thr Lys Glu Ile Glu Gly Asp Ser CysLeu Pro Trp Val Val 420 425 430 Ser Ser Asn Ser Thr Asp Gly Tyr Thr GlySer Gly Asn Thr Pro Gly 435 440 445 Glu Asp His Glu Pro Phe Pro Gly SerLeu Lys Cys Gly Pro Leu Pro 450 455 460 Gln Cys Ala Tyr Ser Met Gly PhePro Ser Glu Ala Ala Ala Ser Met 465 470 475 480 Ala Glu Ala Gly Val ArgPro Gln Asp Arg Ala Asp Glu Arg Gly Ala 485 490 495 Ser Gly Ser Gly SerSer Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn 500 505 510 Val Thr Gly AsnSer Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met 515 520 525 Asn Phe LysGly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln 530 535 540 Glu GlyPro Gly Ser Ala Glu Pro Glu Ser Glu Pro Val Gly Arg Pro 545 550 555 560Val Gln Glu Glu Thr Leu Ala His Arg Asp Ser Phe Ala Gly Thr Ala 565 570575 Pro Arg Phe Pro Asp Val Cys Ala Thr Gly Ala Gly Leu Gln Glu Gln 580585 590 Gly Ala Pro Arg Gln Lys Asp Gly Thr Ser Arg Pro Val Gln Glu Gln595 600 605 Gly Gly Ala Gln Thr Ser Leu His Thr Gln Gly Ser Gly Gln CysAla 610 615 620 Glu 625 20 amino acids amino acid linear protein notprovided 16 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp ValPro 1 5 10 15 Gly Ser Thr Gly 20 5 amino acids amino acid linear proteinnot provided 17 Asp Tyr Lys Asp Glu 5 6 amino acids amino acid linearprotein not provided 18 His His His His His His 5 33 amino acids aminoacid linear protein not provided 19 Arg Met Lys Gln Ile Glu Asp Lys IleGlu Glu Ile Leu Ser Lys Ile 1 5 10 15 Tyr His Ile Glu Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly Glu 20 25 30 Arg

We claim:
 1. A polypeptide selected from the group consisting of: a) apolypeptide having an amino acid sequence of amino acids 234 to 422 ofSEQ ID NO:6, wherein said polypeptide is capable of binding TRAF 6; b) apolypeptide having an amino acid sequence of amino acids 422-616 of SEQID NO:6, wherein said polypeptide is capable of binding a TRAF selectedfrom the group consisting of TRAF 1, TRAF 2, TRAF 3, TRAF 5 and TRAF 6;c) a polypeptide having an amino acid sequence of amino acids 339-422 ofSEQ ID NO:6, wherein said polypeptide is capable of binding TRAF 6; d) apolypeptide having an amino acid sequence of amino acids 545-616 of SEQID NO:6, wherein said polypeptide is capable of binding a TRAF selectedfrom the group consisting of TRAF 1, TRAF 2, TRAF 3, TRAF 5 and TRAF 6;e) a polypeptide having an amino acid sequence of amino acids 339-362 ofSEQ ID NO:6, wherein said polypeptide is capable of binding TRAF 6; f) apolypeptide encoded by a DNA capable of hybridization to a DNA having anucleotide sequence as set forth in SEQ ID NO:5 under stringentconditions, wherein said stringent conditions include hybridizing at6×SSC at 63° C. and washing in 3×SSC at 55° C., and further wherein thepolypeptide is capable of binding a TRAF selected from the groupconsisting of TRAF 1, TRAF 2,TRAF 3, TRAF 5 and TRAF 6; g) fragments ofthe polypeptides of (a), (c) or (e), wherein the fragments are capableof binding TRAF 6; and h) fragments of the polypeptides of (b), (d) or(f), wherein the fragments are capable of binding a TRAF selected fromthe group consisting of TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6.
 2. Apolypeptide having an amino acid sequence at least about 80% identicalto a polypeptide selected from the group consisting of: a) a polypeptidehaving an amino acid sequence of amino acids 234 to 422 SEQ ID NO:6,said polypeptide being capable of binding TRAF6; b) a polypeptide havingan amino acid sequence of amino acids 422-616 of SEQ ID NO:6, saidpolypeptide being capable of binding a TRAF selected from the groupconsisting of TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6; c) a polypeptidehaving an amino acid sequence of amino acids 339-422 of SEQ ID NO;6,said polypeptide being capable of binding TRAF6; d) a polypeptide havingan amino acid sequence of amino acids 545-616 of SEQ ID NO:6, saidpolypeptide being capable of binding a TRAF selected from the groupconsisting of TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6; e) a polypeptidehaving an amino acid sequence of amino acids 339-362 of SEQ ID NO:6,said polypeptide being capable of binding TRAF6; f) a polypeptideencoded by a DNA capable of hybridization to a DNA having a nucleotidesequence as set forth in SEQ ID NO:5 under stringent conditions, whereinsaid stringent conditions include hybridizing at 6×SSC at 63° C. andwashing in 3×SSC at 55° C., said polypeptide being capable of binding aTRAF selected from the group consisting of TRAF1, TRAF2, TRAF3, TRAF5and TRAF6; g) fragments of a polypeptide of (a), (c) or (e), wherein thefragments are capable of binding TRAF 6; and p1 h) fragments of apolypeptide of (b), (d) or (f), wherein the fragments are capable ofbinding a TRAF selected from the group consisting of TRAF1, TRAF2,TRAF3, TRAP5 and TRAF6.
 3. A polypeptide of claim 1 which furthercomprises a peptide selected from the group consisting of animmunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG™ tag, apeptide comprising at least about 6 His residues, a leucine zipper, andcombinations thereof.
 4. A polypeptide according to claim 2 whichfurther comprises a peptide selected from the group consisting of animmunoglobulin Fc domain, an immunoglobulin Fc mutein, a FLAG™ tag, apeptide comprising at least about 6 His residues, a leucine zipper, andcombinations thereof.